Human homeobox gene ventx and macrophage terminal differentiation and activation, compositions and methods thereof

ABSTRACT

The invention relates to a unique formulated pharmaceutical composition and a novel treatment method of intratumoral injection. The pharmaceutical composition of the invention is the saturated ionic solution of sodium ions and calcium ions (the “medicinal ion bomb”). The invention of intratumoral injection with the “medicinal ion bomb” can be used as the first-line treatment in treating cancer and tumor in nine human organs (carcinoma and tumor of the skin and subcutaneous tissue, breast, prostate, thyroid, lung, liver, genital organ, brain and pancreas) and certain other benign diseases (skin and subcutaneous neoplasm, breast fibrocystic change, benign prostatic hyperplasia and thyroid nodules).

PRIORITY CLAIMS AND CROSS REFERENCE TO RELATED APPLICATION

This application is the U.S. national phase of and claim the benefit ofpriority from PCT/US12/67802, filed Dec. 4, 2012, which claims thebenefit of priority from U.S. Provisional Application Ser. No.61/566,655, filed Dec. 4, 2011, the entire content of each of which isincorporated herein by reference in its entirety.

TECHNICAL FIELDS OF THE INVENTION

The invention relates to a unique formulated pharmaceutical compositionand a novel treatment method of intratumoral injection. Thepharmaceutical composition of the invention is a saturated ionicsolution of sodium ions and calcium ions (the “medicinal ion bomb”). Theinvention of intratumoral injection with the “medicinal ion bomb” can beused as the first-line treatment in treating cancers and tumors in atleast nine human organs (e.g., carcinoma and tumor of the skin andsubcutaneous tissue, breast, prostate, thyroid, lung, liver, genitalorgan, brain and pancreas) and at least four categories of benigndiseases (e.g., skin and subcutaneous neoplasm, breast fibrocysticchange, benign prostatic hyperplasia and thyroid nodules).

BACKGROUND OF THE INVENTION

Cancer is the uncontrolled growth of abnormal cells in the body. Canceris often able to invade other tissues from its original location andspread to other parts of the body through blood and lymphatics. Thereare many types of cancer, which may be classified in pathology andclinical diagnosis into carcinoma, sarcoma, leukemia, lymphoma andmyeloma, and malignant tumors of the central nervous system.

Cancer treatment remains a challenging endeavor to both the patient andthe healthcare provider. Current treatments include surgery,chemotherapy, radiotherapy, immunotherapy, biotherapy, laser therapy,cryotherapy, thermotherapy, etc. While significant advancements havebeen made in recent decades in cancer prevention and treatment, cancersurvival rates are still low for many types of cancers. It is estimatedthat in the U.S. alone, there are over 1.5 million new cases of cancerand more than half million of cancer-related deaths in 2011. Thus,cancer remains a major health threat to the public.

In two published reports, a single component of high concentrationsodium chloride was used in treatment of tumors. In one report, Siegleet al. reportedly used hypertonic saline to treat one patient who hadmultiple glomus tumors, a rare benign neoplasm arising from the glomusbody under fingernails and tympanic membrane. (Siegle, et al. 1994 J.Dermatologic Surgery & Oncology 20:347-348.) A single component of 23.4%sodium chloride solution was used, which did not result in successfultreatment of the patient. This patient received 4 sessions ofintralumenal injection with 23.4% hypertonic saline in 6 months. Inanother report, Lin et al. used 36.5% hypertonic saline to treat livertumor in rabbits and none of the tumors were successfully treated. (Lin,et al. 2005 Am. J. Roentgenology 184: 212-219.)

Therefore, an urgent need remains for novel and effective treatments forcancer.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery of uniquepharmaceutical compositions useful for treating various types of cancer,malignant tumor, benign tumor and nonmalignant disease and relatedmethods of use. The present invention addresses the long-felt need forfast and effective treatments that are affordable.

In particular, the invention relates to the development of novelpharmaceutical compositions for treating cancer, malignant tumor, benigntumor and nonmalignant disease. This pharmaceutical composition of theinvention is referred to as the “medicinal ion bomb.” In a preferredembodiment, the composition of the “medicinal ion bomb” comprises 5.0479M of sodium ions (Na⁺) (e.g., from sodium chloride (NaCl)) and 250 mM ofcalcium ions (Ca²⁺) (e.g., from calcium chloride (CaCl₂)) in steriledistilled water. (1 M=1 mol/L; 1 mM=1×10⁻³ M.) In certain preferredembodiments, the composition includes two additional components: 10milliliters (mL) of Ultravist 370 and 20 milligrams (mg) of adrenalineare mixed with per liter of the “medicinal ion bomb” solution beforeintratumoral injection is performed. Herein, both Na⁺ and Ca²⁺ in theformula are utilized to kill cancer cells.

The combination of Na⁺ and Ca²⁺ in the formulation of the “medicinal ionbomb” provides synergetic, complementary and dual-wave cancer-killingeffects. Sterile distilled water is the dissolvent. The Ultravist 370 isan X-ray contrast medium for tracking the diffusion of the “medicinalion bomb” solution inside the treated cancer or malignant tumor undercomputerized tomography (CT) scan guidance. Adrenaline is avasoconstrictive drug to enhance the cancer killing effect by thedelayed release of the “medicinal ion bomb” compositions into bloodcirculation. Not wishing to be bound by the theory, it is believed thatadrenaline can make contraction of blood vessels to create a momentary“artificial tumor capsule” in the treated tumor to prevent the“medicinal ion bomb” solution from flushing out of a non-capsule tumormass.

It is demonstrated herein that the “medicinal ion bomb” is capable ofkilling 18 types of human cancer cell lines in tissue culture withinminutes. The “medicinal ion bomb” also killed human cancer models innude mice within days. Mouse tumors equal to about 2-3% of body weightwere killed by one single intratumoral injection with 0.12 mL of the“medicinal ion bomb.” In selected human patients, a benign tumor orcancer lesion at 50 millimeters (mm)×70 mm in size was killed by onesingle intratumoral injection with the “medicinal ion bomb.” Thisinvention has been successfully applied in 78 human patients whosuffered from 6 types of malignant tumor or cancer and 10 types ofbenign neoplasm or non-malignant conditions.

In one aspect, the invention generally relates to a pharmaceuticalcomposition useful for treating a cancer, malignant tumor, benign tumorand nonmalignant disease via administration by intratumoral injection.The composition includes, in an aqueous solution, a Na⁺ source providinga Na⁺ concentration from about 2.0 M to about 5.5 M and a Ca²⁺ sourceproviding a Ca²⁺ concentration from about 50 mM to about 6.0 M in theaqueous solution. In certain preferred embodiments, the Na⁺ source isNaCl and the Ca²⁺ source is CaCl₂.

In another aspect, the invention generally relates to a compositionuseful for the preparation of a pharmaceutical composition for treatinga tumor, comprising Na⁺ and Ca²⁺ in amounts such that when appropriateamount of water is added thereto produces a pharmaceutical composition(saturated ionic solution) comprising about 5.0479 M of Na⁺ and about250 mM of Ca²⁺ at room temperature.

In yet another aspect, the invention generally relates to a method fortreating a tumor in a patient, comprising intratumoral injection ordirect injection into the tumor lesion a pharmaceutically effectiveamount of a composition comprising about 5.0479 M of Na⁺ and about 250mM of Ca²⁺ at room temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a MCA207 mouse sarcoma model in C57BL6 mouse. FIG. 1A. Apretreatment tumor at 8 mm×8 mm in size. FIG. 1B. The standard procedureof intratumoral injection with the “medicinal ion bomb” in mouse. FIG.1C. The mouse sarcoma was killed by a single injection with 0.10 mL ofthe “medicinal ion bomb.”

FIG. 2 shows a MCF7 human breast cancer model in nude mouse. FIG. 2A. Apretreatment tumor at 13 mm×13 mm in size that is equal to 3%±bodyweight of the mouse. FIG. 2B. The standard procedure of intratumoralinjection with the “medicinal ion bomb” in mouse. FIG. 2C. A large humanbreast cancer model in nude mouse was killed by a single injection with0.14 mL of the “medicinal ion bomb.”

FIG. 3 explains the use of fluorescent Ca²⁺ probe technique in ex vivodetermination of the influx of Ca²⁺ from the extracellular fluid intointracellular fluid in MCF7 human breast cancer cells. FIG. 3A. Sevenpretreatment cancer cells were labeled with fluorescent Ca²⁺ probe. FIG.3B. Two of 7 cancer cells were killed by the “medicinal ion bomb” in14±seconds after starting treatment. FIG. 3C. All 7 cancer cells werekilled in 18±seconds after starting treatment. The procedure ofmeasuring the influx of Na⁺ was the same as the abovementioned but usingthe fluorescent Na⁺ probe.

FIG. 4 shows pictures of the video image from the Nikon Diaphot invertedmicroscope. FIG. 4A. Pretreatment MCF7 human breast cancer cells. InFIG. 4B, the treated cancer cells were experiencing a vortex-likerotation in tissue culture medium in 5 to 7 seconds after adding the“medicinal ion bomb.” FIG. 4C. All cancer cells were killed in16±seconds after starting treatment. The cells in these 3 micrographswere from the same field. Magnification ×400.

FIG. 5 shows a group of micrographs to describe pathologic features ofin vivo MCA207 mouse sarcoma treated with the “medicinal ion bomb.” FIG.5A. White lines surrounding cancer cells are sodium ions (salts) andcalcium ions (salts) in the cancer tissue in mouse. Magnification ×1000.FIG. 5B. The treated cancer tissues were necrotic where the “medicinalion bomb” reached. Magnification ×400. FIG. 5C. Multiple thromboses in atreated tumor section. Magnification ×400.

FIG. 6 shows a group of scanning electron microscope (SEM) pictures ofMCF7 human breast cancer models in nude mice. SEM1 shows the controlcancer cells. SEM2. Membranes of human breast cancer cells were brokeninto pieces and debris. SEM3 discloses severely damaged tumor bloodvessels, particularly damages to the outer covering of arterioles orvenules in tumor tissue. Many tumor vessels were transversely brokenoff.

FIG. 7 shows a group of transmission electron microscope (TEM) picturesof MCF7 human breast cancer models in nude mice. TEM1 was the controlcancer cells. TEM2 reveals heavily damaged cell membrane, nucleus,mitochondria, and cytoplasmic organelles of the cancer cell. TEM3discloses the completely destroyed outer layer, middle layer and theinner layer of tumor vessel. An endothelial cell in the inner layer of avenule comes off. Magnifications ×5000.

FIG. 8 shows a clinical doctor who is performing intratumoral injectionwith the “medicinal ion bomb” in a patient who suffered from a largetumor. FIG. 8A is a pretreatment tumor at 40 mm×60 mm in size in thepatient's right shoulder. FIG. 8B demonstrates a pressure injection with3 mL of the “medicinal ion bomb” to kill the tumor. FIG. 8C indicatesthat the opening of the needle is upward, and in FIG. 8D the opening ofthe needle was downward.

FIG. 9 shows the case of an 82 year-old lady who suffered from a hugesquamous cell carcinoma in the lower back for 3 years. FIG. 9.1. Thepretreatment carcinoma is at 70 mm×70 mm in size. FIG. 9.2. A singleintratumoral injection with 26 mL of the “medicinal ion bomb” solutionwas administered into the cancer lesion. FIG. 9.3. This is a counterpartphoto of the video image from a television apparatus 5 days aftertreatment. Macroscopically, the treated entire cancer lesion wascompletely dead. Microscopically, all treated cancer tissue wasnecrotic.

FIG. 10 shows the case of a 70 year-old lady who suffered from asquamous cell carcinoma in the left face. FIG. 10A. The pretreatmentcancer was 15 mm×15 mm in size. This cancer was killed by one singleintratumoral injection with 1.0 mL of the “medicinal ion bomb” solution.FIG. 10B. The standard procedure of intratumoral injection with the“medicinal ion bomb” in human patient. FIG. 10C. Seven days aftertreatment, the tumor showed coagulative necrosis that looked like acharred dead tumor. The wound was healed without defect by 2 weeks. Thecancer did not return when a 3 year follow-up study was performed aftertreatment.

FIG. 11 shows the case of a 52 year-old lady who suffered from a skinmalignant tumor. This is an uncommon tumor that has a second tumor (theoffspring tumor, brown) growing from its parental tumor (black). FIG.11A. The pretreatment tumor was 20 mm×20 mm in size. FIG. 11B. Thestandard procedure of intratumoral injection with the “medicinal ionbomb” in human patient. FIG. 11C. Two tumors were killed by a singleintratumoral injection with 0.5 mL of the “medicinal ion bomb” in 3 daysand the wound was healed by 2 weeks after treatment. A 3 year follow-upstudy showed no recurrence of the tumor.

FIG. 12 shows the case of a 74 year-old man who suffered from ametastatic cancer in the left inguinal area from the penis. FIG. 12A.The pretreatment cancer was 50 mm×70 mm in size. FIG. 12B. The cancerlesion was treated by one single intratumoral injection with 3.5 mL ofthe “medicinal ion bomb” solution. FIG. 12C. The large cancer in humanpatient was killed by 4 days and the wound was healed by 3 weeks.

FIG. 13 shows the case of an 82 year-old man who suffered from melanomain the front chest for 2 years. FIG. 13A. A typical pretreatmentbutterfly-pattern melanoma in human patient. FIG. 13B. A counterpartphoto of the video image to demonstrate a single intratumoral injectionwith 1.2 mL of the “medicinal ion bomb” to kill this melanoma. FIG. 13C.The melanoma was killed in days and the wound was healed by 3 weeksafter treatment. A 9 year follow-up study demonstrated no recurrence ofmelanoma.

FIG. 14 shows a 58 year-old man who suffered from basal cell carcinomain the front chest. FIG. 14.1 is a pretreatment basal cell carcinoma at12 mm×12 mm in size. FIG. 14.2. The basal cell carcinoma regressed oneweek after a single intratumoral injection with 0.5 mL of the “medicinalion bomb.” FIG. 14.3. The site of the regressed carcinoma was healed by3 weeks after treatment. An 8 year follow-up study showed no recurrenceof the treated basal cell carcinoma.

FIG. 15. FIG. 15.1 shows an image of angiography through hepatic arterythat shows a large liver cancer in the right lobe of the liver in a 62year-old man. FIG. 15.2. The image of the large hepatic carcinoma beforetreatment from the same patient. A long catheter needle was placed inthe center of this large liver cancer and 20 mL of the “medicinal ionbomb” solution was injected. FIG. 15.3. The cancer became smaller fromthe pretreatment 67 mm×67 mm×80 mm to posttreatment 45 mm×45 mm×60 mm insize 7 days after treatment.

FIG. 16 shows the case of a lady who suffered from breast fibrocysticchange in the inventor's clinical study group. FIG. 16.1. An image ofpretreatment mammography shows a large cyst at 40 mm×40 mm in size thatwas located behind of the left nipple and several small fibrocysticlesions were surrounding the large cyst. FIG. 16.2. The mucosa-likefluid inside the cyst was sucked, and then the large cyst is repeatedlywashed and sucked. Then a single intra-cystic injection with 3.5 mL ofthe “medicinal ion bomb” was performed. FIG. 16.3. The image of theposttreatment mammography to demonstrate the fibrocystic breast diseasewas cured 3 months after treatment.

FIG. 17 shows the case of a 78 year-old man who suffered from lipoma.This tumor was located in his rear neck for 18 years. FIG. 17A. Thepretreatment tumor was 40 mm×40 mm in size. FIG. 17B. A singleintratumoral injection with 2.0 mL of the “medicinal ion bomb” wasadministered into the tumor. FIG. 17C. The large tumor was cured by 8weeks. A 9 year follow-up study showed no recurrence of the treatedtumor.

FIG. 18 shows the case of a patient who suffered from atheroma. FIG.18A. A pretreatment atheroma in the vertex was 18 mm×18 mm in size. FIG.18B. A counterpart photo of the video image to demonstrate a singleintratumoral injection with 0.5 mL of the “medicinal ion bomb.” FIG.18C. The atheroma was cured in 2001. This photo was taken when a 9 yearfollow-up study was performed.

FIG. 19 shows the case of a 66 year-old man who suffered from a largeatheroma at 22 mm×28 mm in size. The tumor was treated using a singleintratumoral injection with 1.5 mL of the “medicinal ion bomb” in 2001.A 9 year follow-up study showed no recurrence of the treated tumor.

FIG. 20 shows the application of intralesional injection with the“medicinal ion bomb” in cosmetic conditions. FIG. 20.1 shows a skin molein the patient's face. FIG. 20.2. A single intralesional injection with0.2 mL of the “medicinal ion bomb”. FIG. 20.3. The mole developedcoagulative necrosis by 7 days posttreatment. FIG. 20.4. The skin molewas removed without scar defect.

DETAILED DESCRIPTION OF THE INVENTION

An occasional opportunity sparked this invention. Previously, theinventor used intratumoral injection of arsenic in experimentaltreatment of tumors in mice, which did not work well. One day, theinventor went shopping in a Chinese supermarket and bought a dozensalted duck eggs. On the way back home, he wondered why these saltedduck eggs could be preserved at room temperature for years withoutrotting. The answer is that proteins and yolks in salted duck eggs arecoagulated or clotted by high concentration of salt (sodium chloride),in particular by high concentration of sodium ions. The inventor linkedthis phenomenon to cell biology, cell physiology, membrane potential,ion science and cancer treatment, and presumed that high concentrationsof sodium chloride might coagulate or clot proteins in cancer cells likewhat occurs in salted duck eggs.

A series of experiments were conducted using various solutions of highconcentrations of sodium chloride, calcium chloride, and the “medicinalion bomb” solution having both sodium and calcium ions. Three animaltumor models of MCA207 mouse sarcoma, MC38 mouse colorectal cancer andB16F1 mouse melanoma were established in C57BL6 mice. The treatment ofintratumoral injection started when tumors reached 8 mm×8 mm in size.

Thirty-six C57BL6 tumor mice were randomly divided into three groupsaccording to tumor types (12 animals in each group). Then, animals ineach group were further divided into 4 subgroups (3 tumor mice in eachsubgroup). Tumor animals in subgroup-1, as the control, were treated byintratumoral injection with 0.1 mL of normal saline. Tumor animals insubgroup-2 were treated by intratumoral injection with 0.1 mL of variousconcentrations of sodium chloride solution. Tumor animals in subgroup-3were treated by intratumoral injection with 0.1 mL of variousconcentrations of calcium chloride solution. Tumor animals in subgroup-4were treated with the “medicinal ion bomb” solution.

Twenty-four, 48 and 72 hours after treatment, experimental results wereevaluated by measuring the dimension of necrotic area of the treatedtumors using a ruler. No necrosis of tumors was seen in the controlgroup. However, necrosis occurred in all tumors treated with highconcentrations of sodium chloride solution, calcium chloride solutionand the “medicinal ion bomb” solution. In representative examples, onesingle intratumoral injection with 0.1 mL of 5.0479 M of sodium chloridesolution caused average 90%±5 necrosis of all treated tumors 48 hoursposttreatment. One single intratumoral injection with 0.1 mL of 250 mM,1.0 M, 1.5 M, 2.0 M and 3.0 M of calcium chloride solutions causedcoagulating necrosis of all treated tumors 48 hours posttreatment. Onesingle intratumoral injection with 0.1 mL of the “medicinal ion bomb”solution reached 100% necrosis of all treated tumors 48 hoursposttreatment.

Further, the inventor examined the therapeutic effect of the “medicinalion bomb” on 3 human cancer models in nude mice, including U87 humanglioblastoma, MCF7 human breast cancer and PC3 human prostate cancer.The number of nude mice, grouping of animals, and all experimentalmethods and injection doses in each subgroup were the same as in theC57BL6 mouse tumor models. The data showed that a single intratumoralinjection with 0.1 mL of the “medicinal ion bomb” successfully removedthe tumor in each of all 3 types of human cancer models in nude mice.

Intratumoral injection with the “medicinal ion bomb” was successfullyapplied in treating 78 human patients, including 6 types of malignanttumors or carcinoma (basal cell carcinoma, squamous cell carcinoma,maxillary carcinoma, melanoma, metastatic carcinoma from the penis andliver cancer) and 10 types of benign tumors (atheroma, lipoma, neoplasm,cyst, breast fibrocystic change, lymphadenopathy, thyroid nodule,genital tumor, skin mole, and cosmetics). The “medicinal ion bomb”comprises two active components of Na⁺ and Ca²⁺ or two active compoundsof sodium chloride and calcium chloride. In a preferred embodiment, the“medicinal ion bomb” comprises 5.0479 M of sodium chloride and 250 mM ofcalcium chloride in a solution of sterile distilled water.

In a preferred embodiment, the route of administration of the “medicinalion bomb” is via intratumoral injection (e.g., direct injection) of the“medicinal ion bomb” into tumor or cancer in the subject.

When sodium ion (e.g., NaCl) is used alone for intratumoral injection,the effective treatment concentration of Na⁺ ranges from about 2.0 M toabout 5.4414 M at room temperature. When calcium ion (e.g., CaCl₂) isused alone for intratumoral injection, the effective treatmentconcentration of Ca²⁺ ranges from about 50 mM to 6.0 M at roomtemperature.

It is noted that the Na⁺ source is not limited to NaCl, and the Ca²⁺source is not limited to CaCl₂. Other examples of Na⁺ include inorganicor organic salts, for example, sodium bicarbonate, sodium carbonate, andsodium chlorate, et al. Other sources of Ca²⁺ include inorganic ororganic salts, for example, calcium gluconate, calcium carbonate, andcalcium acetate, et al.

There are a number of differences between the “medicinal ion bomb”compositions herein and two previous reports using a single component ofsodium chloride. (1) This invention is to use ionic Na⁺ and Ca²⁺ incancer treatment. However, previous reports used hypertonic saline. (2)The “medicinal ion bomb” compositions include up to five components inthe specified amounts. The inventor discovered when Na⁺ and Ca²⁺ arecombined in the pharmaceutical formulation the newly formulated“medicinal ion bomb” produces synergetic, complementary and dual-wavecancer-killing effects. (3) In this invention, calcium ion is used asthe “enhancer” of cancer treatment because calcium ion is one of secondmessengers in cell signal transduction biology. Calcium ions alsoinvolve in the metabolism of many enzymes to activate protein kinase Cand assist in the activation of another second messenger cAMP. Calciumions regulate the protein calmodulin to produce an alpha helicalstructure. Therefore, slight increase in the amount of calcium ions inthe intracellular fluid causes tremendous pathophysiological changes inthe internal environment of cells and huge biological transformations inmolecular regulations of cells, which cause either sickness or apoptosisor death of treated cancer cells. (4) The ion concentrations of the“medicinal ion bomb” disclosed herein are formulated on a large amountof experimental data. (5) In this invention, mechanisms of highconcentration Na⁺ and Ca²⁺ to kill cancer cells have been verified fromaspects of cell physiology, molecular biology, cellular membranebiology, membrane potential, ion channels, pathology, scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM). (6) Theinvention herein has for the first time advanced the indications of theintratumoral injection with the “medicinal ion bomb” andprecision-guided intratumoral injection with the “medicinal ion bomb”are indicated for treating cancer, malignant tumor, benign tumor andnonmalignant disease in about nine human organs (carcinoma and tumor ofthe skin and subcutaneous tissue, breast, prostate, thyroid, lung,liver, genital organs, brain and pancreas) and four categories of benigndiseases (skin and subcutaneous neoplasm, breast fibrocystic changes,benign prostatic hyperplasia and thyroid nodules). (7) The preferredroute of administration of the “medicinal ion bomb” is via intratumoralinjection or direct injection with the “medicinal ion bomb” into cancerlesion or tumor condition. (8) It is for the first time to use theconcept and definition of the precision-guided intratumoral injectionwith the “medicinal ion bomb” in the treatment of cancer and otherbenign diseases under the guidance of CT scan or ultrasound imaging. (9)The concentration of Na⁺ in the preferred the “medicinal ion bomb”solution is different from that of two previous reports (e.g., theformula of the claimed invention includes 5.0479 M of Na+ (NaCl) and 250mM of Ca2+ (CaCl2) in sterile distilled water to make the saturatedionic solution). (10) The invention of intratumoral injection with themedicinal ion bomb” has been successfully used in the treatment of 78human patients who suffered from 6 types of cancer and 10 types ofbenign tumors and nonmalignant diseases. Therefore, clinical applicationof the invention in the treatment of types of diseases is different fromthat the previous reported.

As used herein, “benign tumor” refers to all of single or multiple solidtumors or neoplasm in pathologic classification including, but notlimited to, adenoma, angioma, atheroma, fibroma, lipoma, teratoma,thyroma, cyst, polyp, skin mole, tag and wart, and other neoplasmconditions in human patients.

As used herein, “malignant tumor” refers to all of single or multiplesolid malignancies in pathologic classification including, but notlimited to, cancer, carcinoma, lymphoma, melanoma, myeloma, sarcoma,brain tumors and other malignant tumors in human patients.

Cell Physiology in Development of the “Medicinal Ion Bomb”

In cell physiology, survival of normal cells, tumor cells or cancercells is dependent upon the equilibrium of osmotic pressure inside andoutside of cells. Normal osmotic pressure of cells depends on theequilibrium of concentrations of ions between the extracellular andintracellular fluids. Physiologically, Na⁺, K⁺ and Ca²⁺ play importantroles in maintenance of living cells. When concentrations of ionsbetween the extracellular and intracellular fluids are identical, it iscalled isotonic. Since the cell is at equilibrium, there is no ionicconcentration gradient, and the flow of water in is equal to the flow ofwater out. This does not cause sickness or death of the cell. When acell has a higher ionic concentration inside the cell than out, it ishypertonic. This causes a net flow of water into the cell. When a cellhas a lower ionic concentration inside the cell than out, it ishypotonic and water flows out of the cell. (Costanzo L S, 2010 CostanzoPhysiology, 4^(th) edition, Saunders, Philadelphia)

Without intending to be so limited to this principle, it is believedthat a significant change in concentrations of Na⁺ and Ca²⁺ between theextracellular and intracellular fluids causes tremendous transformationsin physiology of cells that result in either sickness, apoptosis ordeath of cells. The “medicinal ion bomb” is intentionally designed tohave high concentrations of Na⁺ and Ca²⁺ to kill cancer cell lines intissue culture, cancer models in animals and large carcinomas in humanpatients.

One strategy is to deliver high concentrations of Na⁺ and Ca²⁺ to theextracellular fluid or intratumoral tissue where both ions can rapidlycross the membranes of cells to the intracellular fluid.

First, potential roles of Na⁺, K⁺, Ca²⁺ and Mg²⁺ in treatment of tumorand cancer were tested. In vivo experiments in BALB/c mice wereperformed. High concentration solutions of Na⁺, K⁺, Ca²⁺ and Mg²⁺ wereinjected intramuscularly. However, high concentrations of K⁺ and Mg²⁺solutions are toxic and caused paralysis of legs, failure ofcardiovascular and pulmonary systems, and death of animals. Experimentswere focused on high concentrations of Na⁺ and Ca²⁺ solutions.

Different concentrations of sodium chloride solution were prepared, andthe test started from 1.0 M, 1.5 M, 1.711 M, 3.0 M, 4.28 M, 4.449 M,5.0479 M, 5.133 M to 5.4414 M. Various concentrations of calciumchloride solution were prepared, and the test started from 25 mM, 50 mM,100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, 901 mM, and 1.0M, 1.5 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 5.5 M to 6.0 M.

After comparative studies, the formula of the preferred “medicinal ionbomb” was found to be 5.0479 M of Na⁺ and 250 mM of Ca²⁺ in sterilewater, which is the saturated ionic solution of the combination of Na⁺and Ca²⁺ at room temperature.

There are at least three rationales for choosing Na⁺ to be the firstactive component of the “medicinal ion bomb.” (1) Na⁺ is an essentialand safe ion and its concentration in the body is very flexible.Medicinal sodium chloride solutions have been long-term used forintravenous infusion in human patients. There is no evidence to showthat Na⁺ is toxic or a mutagenic agent when used in properconcentrations. (2) Na⁺ has a unique feature of fast diffusion in livingtissue and tumor tissue. As shown under the monitor of the real-timeX-ray imaging in this invention, the diffusion of 5.0479 M of sodiumchloride solution is approximately 25-fold faster than 1.0 M and 2.0 Mof calcium chloride solution in human cancer model in nude mice. (3) Asindicated, high concentrations of sodium chloride solutions are capableof killing multiple types of animal tumors in C57BL6 mice and humancancer models in nude mice.

In this study, Ca²⁺ is used as the second active component of the“medicinal ion bomb.” Ca²⁺ is one of second messengers in cell signaltransduction biology and its intracellular concentration is less than0.0001 mM. We confirmed that the tissue culture medium containing 250 mMof Ca²⁺ induced sickness, apoptosis and death of 18 types of humancancer cell lines. Fluorescence microscopy using the fluorescent Ca²⁺probe demonstrated that 250 mM, 1.0 M, 1.5 M, 2.0 M and 3.0 M of Ca²⁺solutions caused immediate sickness or death of cancer cells.

Ex Vivo Killing Test of the “Medicinal Ion Bomb”

Roles of various concentrations of Na⁺ and Ca²⁺ solutions and thepreferred “medicinal ion bomb” solution were tested. Tumor cells werecultured in DMEM or RPMI1640 medium supplemented with 10% fetal bovineserum (FCS). The medium in the control group was free of the “medicinalion bomb” solution. The “medicinal ion bomb” solution was added in all18 experimental cancer groups. Mortality of cancer cells was evaluatedby Trypan blue exclusion test or flow cytometry. The data showed thatthe “medicinal ion bomb” killed all 18 types of human cancer cell linesin tissue culture in 3 to 30 minutes, including A549 human lung cancer,ARO human thyroid cancer, CWR-22 human prostate cancer, HeLa humancervical cancer, HL60 human leukemia, HT-29 human colorectal cancer,Jurkat human T cell leukemia, K562 human leukemia, M2 human lymphoma,M24 Human melanoma, MCF7 human breast cancer, MDA-MB-231 human breastadenocarcinoma, OVCAR3 human ovary cancer, PC3 human prostate cancer,SK-RC-52 human kidney cancer, T-47D human breast epithelial tumor, LNCaphuman prostate cancer, and U87 human glioblastoma.

Minimal and Maximal Effective Treatment Concentrations of Na⁺ and Ca²⁺

The in vivo treatment effect of the “medicinal ion bomb” was tested in 3mouse tumor models. High concentrations of sodium chloride solution,calcium chloride solution, and the preferred “medicinal ion bomb”solution were injected into MCA207 mouse sarcoma, MC38 mouse colorectalcancer and B16F1 mouse melanoma in C57BL6 mice. The data showed thatwhen Na⁺ solution was used alone for intratumoral injection its minimaleffective treatment concentration is 2.0 M at room temperature. The 3 Mconcentration of Na⁺ solution caused average 30%±10 necrosis of alltreated tumors in 48 hours. One single intratumoral injection with5.0479 M of Na⁺ solution caused average 90%±5 necrosis of all treatedtumors in 48 hours. The maximal effective treatment concentration of Na⁺(e.g., sodium chloride) solution was 5.4414 M at room temperature thatcaused average 92%±5 necrosis of all treated tumors in 48 hours. Forintratumoral injection or direct injection into a tumor, the minimaleffective treatment concentration of Ca²⁺ (e.g., calcium chloride)solution was 50 mM at room temperature, and the maximal effectivetreatment concentration was 6.0 M at room temperature when it was usedalone for intratumoral injection. However, high concentration of calciumchloride solution is toxic and causes unhealed wound at the injectionsite.

Diffusion of the “Medicinal Ion Bomb” in Tumor Tissue

Ex vivo diffusion experiments were performed in fresh beef liver at roomtemperature. Thirty parts of the “medicinal ion bomb” solution was mixedwith one part of India ink. The mixed “medicinal ion bomb” solution wasinjected into beef livers starting from 1.0 mL, 2.0 mL and 3.0 mL. Sixtyminutes after injection, beef livers were cut and dimensions ofdiffusion areas were measured with a ruler.

In vivo diffusion experiments were conducted in mice under the guidanceof X-ray imaging. Ninety-nine mL of the “medicinal ion bomb” solutionmixed with one mL of the X-ray contrast enhancer Ultravist 370, and 0.12mL of the mixed “medicinal ion bomb” solution was injected into a humancancer model in nude mouse. Normally, a mouse tumor or human cancermodel at 8 mm×8 mm to 10 mm×10 mm in size is fully filled with 0.12 mLof the mixed “medicinal ion bomb” solution in 3 to 5 minutes. The datademonstrated that the diffusion time and diffusion velocity of 5.0479 Mof sodium chloride solution was approximately 25-fold faster than 1.0 Mand 2.0 M of calcium chloride solution in tumor tissue in living mice.

Findings of the different diffusion time, different diffusion velocityand different diffusion dimension resulting from Na⁺ and Ca²⁺ solutionsinspired the inventor to create the formula of the “medicinal ion bomb.”The key idea of this invention is to develop a powerful “medicinal ionbomb” for treating cancer, malignant tumor, benign tumor andnonmalignant disease by the combination of Na⁺ and Ca²⁺. Because thediffusion of Na⁺ is fast and the diffusion of Ca²⁺ is slow in tumortissue, and because both types of ions are capable of killing cancerswhen they are used separately, we postulated that the combination of Na⁺and Ca²⁺ in the formula of the “medicinal ion bomb” will generatesynergetic, complementary and dual-wave cancer-killing effects. Thefirst wave of in vivo cancer-killing effect (the early killing action)is mediated by Na⁺, which happens in one to 12 hours after treatment.The second wave of in vivo cancer-killing action (the later killingeffect) is mediated by Ca²⁺, which occurs in 6 to 24 hours aftertreatment. This hypothesis has now been proven by pathologic findingsdisclosed herein.

Relationship Between Influx of Na⁺—Ca²⁺ and Death of Cancer Cells

Three tracking techniques of ion channels were utilized to explain therelationship between influx of Na⁺ and Ca²⁺ from the extracellular fluidinto intracellular fluid and death of cancer cells. The patch clamptechnique is a commonly used method to measure the membrane potential ofcardiomyocytes. Here, the patch clamp technique was used to demonstratethe relationship between changes in the membrane potential and death ofmouse cardiomyocytes. Normal mouse cardiomyocytes were pre-cultured inthe patch clamp chamber and membrane potentials were measured before andafter adding the “medicinal ion bomb” solution. The membrane potentialof mouse cardiomyocytes was automatically recorded. When 5% and 10% ofthe “medicinal ion bomb” media were added in the patch clamp chamber,mouse cardiomyocytes died in 5 and 3 minutes, respectively. When 100% ofthe “medicinal ion bomb” solution was added in the patch clamp chamber,mouse cardiomyocytes died in dozen seconds. The data indicated thatcell-killing action of the “medicinal ion bomb” is a dose- andtime-dependence.

Fluorescent ion probe technique was used in study of the influx of Na⁺and Ca²⁺ from the extracellular fluid into intracellular fluid of cancercells. Before and after adding the “medicinal ion bomb” solution to thecell chamber, fluorescence image and time of the influx of Na⁺ or Ca²⁺probe into cancer cells were real-time recorded. If a cancer cell iskilled, the fluorescence image of the cancer cell labeled with Na⁺ orCa²⁺ probe disappears from the computer screen, indicating that eitherthe membrane of cancer cell is broken up or the entire cancer cell isbusted. The fluorescent Na⁺ probe image data showed that use of 5.0479 Mof Na⁺ solution alone took 20±4 seconds to kill MCF7 human breast cancercells. When the preferred “medicinal ion bomb” solution was used it onlytook 18±4 seconds to kill the same type of cancer cells, indicating thatCa²⁺ has an enhancer effect in cancer treatment, and indicating that theformula of the combination of Na⁺ and Ca²⁺ has synergetic andcomplementary cancer-killing effect.

The Nikon Diaphot inverted microscopy is a real-time imaging system forinvestigation of living cells. It is equipped with a video imagingapparatus and a photograph unit. The video data revealed 5 pathologicphases of the “medicinal ion bomb” to kill cancer cells: (1) Rapidcellular membrane disruption of cancer cells; (2) acute dehydration ofcancer cells because of high concentrations of Na⁺ and Ca²⁺ in theextracellular fluid after adding the “medicinal ion bomb;” (3) swellingof cancer cells due to the influx of large amount of Na⁺ and Ca²⁺ fromthe extracellular fluid into intracellular fluid; (4) busting of cancercells caused by extremely swollen cancer cells; and (5) immediate deathof cancer cells. The entire process of 5 pathologic damages to cancercells occurred in dozen seconds and each of the pathologic phasesoccurred in only 3 to 4 seconds.

Under physiological conditions, the concentration of Na⁺ in theextracellular fluid is 140 mM and intracellular fluid is 14 mM. Becausethe preferred “medicinal ion bomb” solution has 5.0479 M of Na⁺, theconcentration of Na⁺ in the extracellular fluid of cancer cells is36-fold higher than its normal level and the concentration of Na⁺ in theintracellular fluid is about 360-fold higher than its normalconcentration. As a result, cancer cells are killed in dozen seconds byextremely high concentration of Na⁺ from the “medicinal ion bomb.”

On the other hand, the normal concentration of Ca²⁺ in the extracellularfluid is 2.5 mM and intracellular fluid is 0.0001 mM in mammalian cells.The “medicinal ion bomb” contains 250 mM of Ca²⁺, which is 100-foldhigher than its physiological level in the extracellular fluid andapproximately 2.5 million-fold higher than its physiologicalconcentration in the intracellular fluid. Because Ca²⁺ is one ofsecondary messengers in cell signal transduction biology, highconcentration of Ca²⁺ in the intracellular fluid not only causestremendous physiological damages, but also causes huge biologicalinjuries to cancer cells. Therefore, high concentration of Ca²⁺ in the“medicinal ion bomb” is the cancer-killing enhancer and it canaccelerate death of cancer cells.

Pathological and Ultrastructural Studies

Twenty-four hours after treatment with the “medicinal ion bomb,”specimens for microscopy, SEM and TEM were taken from nude mouse bearingMCF7 human breast cancer or patients whose cancer was treated with the“medicinal ion bomb.” Slides for pathologic examination were stainedwith hematoxylin and eosin and specimens for SEM and TEM were preparedaccording to the corresponding instructions.

Representative pathologic features of human cancer treated with the“medicinal ion bomb” include (1) sodium ions (salts) and calcium ions(salts) infiltrated in surrounding tissue of cancer cells; (2) necrosisof tumor tissues and cancer cells where the “medicinal ion bomb”solution reached, indicating that fully filling a tumor with the“medicinal ion bomb” solution is a necessary condition to kill or cure acancer; (3) thromboses in tumor blood vessels; and (4) massive bleedingin necrotic areas resulting from injured tumor blood vasculatures.

SEM demonstrated that human breast cancer models treated with the“medicinal ion bomb” showed destroyed cancer cell membranes withinnumerous micro-holes. Membranes of cancer cells were broken to piecesor debris. Outer layer tissues of tumor blood vessels came off.Transverse ruptures of arterioles or venules in tumor tissue were seen.TEM showed destructive cell membranes, mitochondria, lysosome, andnucleus of cancer cells. Endothelial cells in the inner lining of tumorvessels were split off from the vascular lumen. Most of cancer cellswere ruptured into several parts and interstitial tissue of the treatedcancer was smashed. All these pathologic and ultrastructural features oftreated mouse tumors and human cancer models looked like the site ofpost-nuclear bombing.

Application of the “Medicinal Ion Bomb” in Human Patients

Amongst 78 human patients, 11 patients suffered from malignant tumors orcancer, including 2 cases of skin basal cell carcinoma, 3 cases of skinsquamous cell carcinoma, 2 cases of melanoma, 1 case of maxillaryadenocarcinoma, 1 case of metastatic cancer from the penis, and 2 casesof liver cancer. With the exception of one patient who suffered fromterminal stage of maxillary carcinoma died in 3 months after treatment,other 10 cancer patients were successfully treated by one singleintratumoral injection with the “medicinal ion bomb.” Other 67 patientswho suffered from 10 types of benign tumors and nonmalignant diseases (1case of genital neoplasm, 1 case of thyroid nodule, 2 cases oflymphadenopathy, 3 cases of skin mole, 4 cases of cysts, 4 cases ofbreast fibrocystic changes, 6 cases of cosmetic conditions, 10 cases ofneoplasm, 15 cases of atheroma and 21 cases of lipoma) were successfullytreated.

When a benign tumor or cancer condition is located in the surface of thebody or subcutaneous tissue, the tumor can be treated under direct viewand no anesthesia is used. Patient is brought to treatment table. Afterdisinfection of the tumor site with 70% alcohol, a 19-gauge needle isplaced into the center of tumor. The tumor or cancer lesion is treatedby intratumoral injection with the preferred “medicinal ion bomb”solution. Special caution is taken not to punch through the oppositecapsule of tumor. A proper injection pressure is maintained for 3 to 5minutes to ensure the entire tumor is fully filled with the “medicinalion bomb” solution. Generally, patient is uneventful throughout thecourse of treatment. Follow-up studies were made in 7, 14, 21 and 28days after treatment.

If a malignant tumor or cancer lesion is located in deep tissue of thebody, for example, brain tumor or pancreatic cancer, a generalanesthesia is used. The precision-guided intratumoral injection with thepreferred “medicinal ion bomb” is performed under CT scan guidance tomonitor if the entire tumor is fully filled with the “medicinal ionbomb” mixed with the Ultravist 370. After treatment, patient ishospitalized for days and follow-up studies are conducted in 7, 14, 21and 28 days by CT scan to determine if the treated cancer lesion hasreduced in size or completely killed.

Needles used for intratumoral injection are from 19 gauge to 26 gauge insize, and extra long needles are used for tumors in deep tissue,depending on the size and location of tumor. Needles and extra longneedles can be bent in any angle to reach a tumor or cancer lesion wherea surgical access is unavailable or a cancer condition is inoperable.

A unique advantage of this invention is that intratumoral injection withthe “medicinal ion bomb” can be used as the first-line treatment ofcancer patients who may be cured or effectively treated without need forsurgery, chemotherapy, and/or radiotherapy. On the other hand, thetreatment provided herein may be combined with surgery, chemotherapy,radiotherapy, immunotherapy, biotherapy and other cancer treatments toenhance the therapeutic result of cancer treatment.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and methods disclosedherein can be administered to cancer patient to kill a cancer lesion insitu before surgery. Then, a “dead cancer” is safely removed by surgery.Advantages of this strategy are to reduce risks of iatrogenic cancermetastases or intraoperative cancer tissue spreading from surgicalprocedure, which increase patient survival rate and tumor curability.

The pharmaceutical compositions of Na⁺ and Ca²⁺ of the invention, whenproperly administered as disclosed herein, are not believed to causeside effect, such as inflammation, pyretogenesis, carcinogenesis, andmutagenesis.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofintratumoral injection with the “medicinal ion bomb” in the inventionmay be used as the first-line treatment in patients suffering fromcutaneous and subcutaneous cancers and malignant tumors including, butnot limited to, basal cell carcinoma, melanoma, sarcoma, skin cancer,and other skin malignant conditions. They may also be used as thefirst-line treatment in patients suffering from cutaneous andsubcutaneous benign tumors including, but not limited to, adenoma,atheroma, hemangioma, lipoma, skin mole, tag and wart, and otherneoplasm conditions.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from brain tumors including, but not limited to, glioma,adenoma and other brain tumors. In selected patients suffering frombrain tumors, the pharmaceutical compositions and treatment methods ofthis invention provide patients an opportunity of possible cure withoutundergoing a high-risk brain surgery for the removal of brain tumor.Patients suffering from glioma and other types of brain tumors onlyundergo a minor surgery to make a small hole in cranial bone for placinga longer needle in a brain tumor under CT scan guidance. This treatmentcan greatly decrease patient mortality, and increase patient survivalrate and tumor curability.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from thyroid cancer. Such patients may avoid a high-risksurgery or isotopic radiotherapy. This treatment can protect patientfrom damage to parathyroid glands from surgical procedure. In addition,it can also be used as a preventive cancer treatment in patients whohave been planned to have a total thyroidectomy or subtotalthyroidectomy. Prior to surgery, for example, thyroid cancer lesion canbe killed in situ by intratumoral injection with the “medicinal ionbomb,” and then a “dead thyroid cancer” is safely removed by surgery.This strategy may protect patient from iatrogenic metastasis orintraoperative cancer tissue spreading, and increase patient survivalrate and tumor curability.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from benign tumors of thyroid glands including, for example,adenoma, thyroma, cyst and nodules in thyroid glands.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from primary or recurrent hyperthyroidism and patients canavoid high-risk isotopic radiotherapy or surgery. Prior to treatment,patient takes iodine by mouth. When patient's thyroid glands becomesmall, intra-thyroid injection with the “medicinal ion bomb” in boththyroid glands is performed. Patient is simply treated by performing alongitudinal injection in each thyroid gland under the guidance ofultrasound imager or CT scan. The mechanism of using the “medicinal ionbomb” in treatment of hyperthyroidism is to reduce the number of thyroidhormone-producing cells as do by surgery and isotopic radiotherapy.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from breast cancer. In selected breast cancer patients, thisinvention provides an opportunity of possible cure using a non-surgicalintervention. Such patients can avoid a destructive surgery. Theintratumoral injection with the “medicinal ion bomb” in this inventioncan also be used as a preventive cancer treatment in patients sufferingfrom primary breast cancer who have been considered for lumpectomy ormastectomy or radical mastectomy. Two to 3 days prior to surgery,patient's breast cancer lesion is killed in situ by intratumoralinjection with the “medicinal ion bomb,” then a “dead breast cancer” issafely removed by surgery. More importantly, it can protect patient fromiatrogenic metastasis from surgery, and increase patient survival rateand cancer curability. Patient is brought to treatment table and istreated with a local anesthesia. Ninety-nine mL of the “medicinal ionbomb” solution is mixed with one mL of the Ultravist 370 medium and thenthe mixed “medicinal ion bomb” solution is directly injected into breastcancer lesion under the guidance of CT scan or ultrasound imager.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from mammary benign diseases including, but not limited to,adenoma, fibroma, fibrocystic change and hyperplasia of breast.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from primary prostate cancer. In selected patients, thisinvention provides an opportunity of possible cure without undergoingconventional hormone therapy, brachytherapy, surgery or chemotherapythat have serious complications, particularly in posttreatment erectiledysfunction and cancer recurrence. The normal prostate is about 17 gramsto 25 grams in weighing. The precision-guided intra-prostatic injectionwith the “medicinal ion bomb” is a minimally invasive treatment thatdoesn't cause the above complications or side effects. Patients can besafely treated through transurethral injection under the guidance of CTscan, ultrasound or cystoscope. According to the clinical experience intreating other cancer conditions in human patients, it only needs about1.0 mL to 2.0 mL of the “medicinal ion bomb” solution to kill a prostatecancer lesion in a human patient.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention is particularly indicated for patients who suffers frombenign prostatic hyperplasia (BPH). In selected patients, this inventionprovides a unique opportunity to cure BPH without undergoing surgery orhormone therapy. One patient who suffered from BPH in the clinical studyof the invention whose condition obtained significant improvement byusing intra-prostatic injection with the “medicinal ion bomb.”

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from primary pancreatic cancer. In selected pancreatic cancerpatients, this invention provides an opportunity of possible cure ofpancreatic cancer without undergoing surgical intervention. Pancreas isan elongated, tapered organ located across the back of the abdomenbehind the stomach. Patient's abdominal organs are not damaged if theinjection access is selected in the posterior back. Patient is placedthe prone position and intratumoral injection with the “medicinal ionbomb” is performed under CT scan guidance. Ninety-nine mL of the“medicinal ion bomb” solution is mixed with one mL of the Ultravist 370medium and then the mixed “medicinal ion bomb” solution is injected intopancreatic cancer through a longer needle or an extra long needle underCT scan guidance.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as a preventive cancer treatment in pancreaticcancer patients who have been considered a surgery. Two to 3 days priorto surgery, a pancreatic cancer lesion is killed in situ by intratumoralinjection with the “medicinal ion bomb,” then a “dead pancreatic cancer”is safely removed by surgery. This strategy can protect patient fromiatrogenic metastasis and increase patient survival rate and cancercurability.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from benign tumors of pancreas such as insulinoma, cyst,adenoma and other pancreatic tumors.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as a palliative treatment of pancreatic cancerduring a surgery when an advanced stage of pancreatic cancer isun-removable. During laparotomy, the “medicinal ion bomb” solution isinjected into pancreatic cancer in direct view. This treatment canrelieve patient's cancer condition and improve the life quality ofpatient.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from primary lung cancer, liver cancer and kidney cancer. Thetreatment is performed under CT scan guidance or direct view duringsurgery.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as a palliative treatment in patientssuffering from colorectal carcinoma and is associated with large bowelobstruction. Under the guidance of endoscope the “medicinal ion bomb” isinjected into cancer lesion in direct view to relieve obstructivesymptoms in emergency.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from cervical cancer. The patient is treated by a simpleintratumoral injection with the “medicinal ion bomb” in direct view.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from pre-malignancy and benign tumors in female and malegenital organs in direct view.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from leiomyoma or leiomyosarcoma in uterus, which can betreated trans-vaginally or under the guidance of peritoneal endoscopy orminor invasive surgery.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used as the first-line treatment in patientssuffering from benign and malignant tumors of ovary including, but notlimited to, ovarian carcinoma, teratoma and fibroid conditions. Patientis treated by placing a longer needle in tumor under the guidance oflaparoscopy or minor invasive surgery.

The pharmaceutical compositions of Na⁺ and Ca²⁺ and treatment methods ofthe invention may be used in cosmetic therapy to remove a skin mole,tag, wart and neoplasm in the surface of the body.

Taken together, the invention of intratumoral injection with the“medicinal ion bomb” can be used as the first-line treatment in treatingcancer and tumor at least in the nine human organs (carcinoma and tumorof the skin and subcutaneous tissue, breast, prostate, thyroid, lung,liver, genital organ, brain and pancreas) and four category benigndiseases (skin and subcutaneous neoplasm, breast fibrocystic changes,benign prostatic hyperplasia and thyroid nodules).

EXAMPLES

This invention is more specifically illustrated by the followingrepresentative examples of the procedure of intratumoral injection withthe “medicinal ion bomb” in animal tumors and human cancer conditions.

The formula of the “medicinal ion bomb” for cancer treatment comprises5.0479 M of Na⁺ and 250 mM of Ca²⁺ plus about 890 mL of steriledistilled water to make the final volume of one liter of the “medicinalion bomb” solution. Two additional compositions include 20 micrograms(μg)/mL of adrenaline and 10 microliters (μL)/mL of Ultravist 370 X-raycontrast medium.

Na⁺ and Ca²⁺ in this formula make up the killing power, distilled wateris the dissolvent, Ultravist 370 is an X-ray contrast medium, andadrenaline is a vasoconstrictor. Studies have confirmed that the formulaof the “medicinal ion bomb” solution using 5.0479 M of NaCl and 250 mMof CaCl₂ at room temperature is a completely dissolved and saturatedionic solution with maximally effective cancer-killing action.

The treatment method of intratumoral injection with the “medicinal ionbomb” solution in the invention has claimed that the route ofadministration of the “medicinal ion bomb” solution is throughintratumoral injection (direct injection) of the “medicinal ion bomb”solution into tumor or cancer in the subject.

In addition, minimal and maximal effective treatment concentrations ofNa⁺ and Ca²⁺ for intratumoral injection have been claimed. When Na⁺(e.g., NaCl) is used alone for intratumoral injection, the effectivetreatment concentration of Na⁺ ranges from about 2.0 M to about 5.4414 Mat room temperature. When Ca²⁺ (e.g., CaCl₂) is used alone forintratumoral injection, the effective treatment concentration of Ca²⁺ranges from about 50 mM to 6.0 M at room temperature.

Example 1

FIG. 1 is a MCA207 mouse sarcoma model in C57BL6 mouse. FIG. 1A. Apretreatment tumor was 8 mm×8 mm in size. FIG. 1B. The standardprocedure of intratumoral injection with the “medicinal ion bomb” inmouse. FIG. 1C. The mouse sarcoma was killed by a single injection with0.10 mL of the “medicinal ion bomb.”

Example 2

FIG. 2 is a MCF7 human breast cancer model in nude mouse. FIG. 2A. Apretreatment tumor was 13 mm×13 mm in size that is equal to 3%±bodyweight of the mouse. FIG. 2B. The standard procedure of intratumoralinjection with the “medicinal ion bomb” in mouse. FIG. 2C. A large humanbreast cancer model in nude mouse was killed by a single injection with0.14 mL of the “medicinal ion bomb.”

Example 3

FIG. 3 shows the use of fluorescent Ca²⁺ probe technique in ex vivodetermination of the influx of Ca²⁺ from the extracellular fluid intointracellular fluid in MCF7 human breast cancer cells. FIG. 3A. Sevenpretreatment cancer cells were labeled with fluorescent Ca²⁺ probe. FIG.3B. Two of 7 cancer cells were killed by the “medicinal ion bomb” in14±seconds after starting treatment. FIG. 3C. All 7 cancer cells werekilled in 18±seconds after starting treatment. The procedure ofmeasuring the influx of Na⁺ was the same as the abovementioned but usingthe fluorescent Na⁺ probe.

Example 4

FIG. 4 shows a group-picture of the video image from the Nikon Diaphotinverted microscope. FIG. 4A. Pretreatment MCF7 human breast cancercells. In FIG. 4B, the treated cancer cells were experiencing avortex-like rotation in tissue culture medium in 5 to 7 seconds afteradding the “medicinal ion bomb.” FIG. 4C. All cancer cells were killedin 16±seconds after starting treatment. The cells in these 3 micrographswere from the same field. Magnification ×400.

Example 5

FIG. 5 shows a group of micrographs to describe pathologic features ofin vivo MCA207 mouse sarcoma treated with the “medicinal ion bomb.” FIG.5A. White lines surrounding cancer cells were sodium ions (salts) andcalcium ions (salts) in the cancer tissue in mouse. Magnification ×1000.FIG. 5B. The treated cancer tissues were necrotic where the “medicinalion bomb” reached. Magnification ×400. FIG. 5C. Multiple thromboses in atreated tumor section. Magnification ×400.

Example 6

FIG. 6 shows a group of SEM of MCF7 human breast cancer models in nudemice. SEM1 shows the control cancer cells. SEM2. Membranes of humanbreast cancer cells were broken into pieces and debris. SEM3 disclosesseverely damaged tumor blood vessels, particularly damages to the outercovering of arterioles or venules in tumor tissue. Many tumor vesselswere transversely broken off.

Example 7

FIG. 7 shows a group of TEM of MCF7 human breast cancer models in nudemice. TEM1 is the control cancer cells. TEM2 reveals heavily damagedcell membrane, nucleus, mitochondria, and cytoplasmic organelles of thecancer cell. TEM3 discloses the completely destroyed outer layer, middlelayer and the inner layer of tumor vessel. An endothelial cell in theinner layer of a venule came off. Magnifications ×5000.

Example 8

FIG. 8 shows a clinical doctor who is performing intratumoral injectionwith the “medicinal ion bomb” in a patient who suffered from a largetumor. FIG. 8A is a pretreatment tumor at 40 mm×60 mm in size in thepatient's right shoulder. FIG. 8B demonstrates a pressure injection with3 mL of the “medicinal ion bomb” to kill the tumor. FIG. 8C indicatesthat the opening of the needle is upward, and in FIG. 8D the opening ofthe needle was downward.

Example 9

FIG. 9 shows the case of an 82 year-old lady who suffered from a hugesquamous cell carcinoma in the lower back for 3 years. FIG. 9.1. Thepretreatment carcinoma was 70 mm×70 mm in size. FIG. 9.2. A singleintratumoral injection with 26 mL of the “medicinal ion bomb” solutionwas administered into the cancer lesion. FIG. 9.3. This is a counterpartphoto of the video image from a television apparatus 5 days aftertreatment. Macroscopically, the treated entire cancer lesion wascompletely dead. Microscopically, all treated cancer tissue wasnecrotic.

Example 10

FIG. 10 shows the case of a 70 year-old lady who suffered from asquamous cell carcinoma in the left face. FIG. 10A. The pretreatmentcancer was 15 mm×15 mm in size. This cancer was killed by one singleintratumoral injection with 1.0 mL of the “medicinal ion bomb” solution.FIG. 10B. The standard procedure of intratumoral injection with the“medicinal ion bomb” in human patient. FIG. 10C. Seven days aftertreatment, the tumor has shown coagulative necrosis that looked like acharred dead tumor. The wound was healed without defect by 2 weeks. Thecancer did not return when a 3 year follow-up study was performed aftertreatment.

Example 11

FIG. 11 shows the case of a 52 year-old lady who suffered from a skinmalignant tumor. This is an uncommon tumor that has a second tumor (theoffspring tumor, brown) growing from its parental tumor (black). FIG.11A. The pretreatment tumor at 20 mm×20 mm in size. FIG. 11B. Thestandard procedure of intratumoral injection with the “medicinal ionbomb” in human patient. FIG. 11C. Two tumors were killed by a singleintratumoral injection with 0.5 mL of the “medicinal ion bomb” in 3 daysand the wound was healed by 2 weeks after treatment. A 3 year follow-upstudy showed no recurrence of the tumor.

Example 12

FIG. 12 shows the case of a 74 year-old man who suffered from ametastatic cancer in the left inguinal area from the penis. FIG. 12A.The pretreatment cancer at 50 mm×70 mm in size. FIG. 12B. The cancerlesion was treated by one single intratumoral injection with 3.5 mL ofthe “medicinal ion bomb” solution. FIG. 12C. The large cancer in thepatient was killed by 4 days and the wound was healed by 3 weeks.

Example 13

FIG. 13 shows the case of an 82 year-old man who suffered from melanomain the front chest for 2 years. FIG. 13A. A typical pretreatmentbutterfly-pattern melanoma in the patient. FIG. 13B. A counterpart photoof the video image to demonstrate a single intratumoral injection with1.2 mL of the “medicinal ion bomb” to kill this melanoma. FIG. 13C. Themelanoma was killed in days and the wound was healed by 3 weeks aftertreatment. A 9 year follow-up study demonstrated no recurrence ofmelanoma.

Example 14

FIG. 14 shows the case of a 58 year-old man who suffered from basal cellcarcinoma in the front chest. FIG. 14.1 was a pretreatment basal cellcarcinoma at 12 mm×12 mm in size. FIG. 14.2. The basal cell carcinomaregressed one week after a single intratumoral injection with 0.5 mL ofthe “medicinal ion bomb.” FIG. 14.3. The site of the regressed carcinomawas healed by 3 weeks after treatment. An 8 year follow-up study showedno recurrence of the treated basal cell carcinoma.

Example 15

FIG. 15.1. An image of angiography through hepatic artery shows a largeliver cancer in the right lobe of the liver in a 62 year-old man. FIG.15.2. The image of the large hepatic carcinoma before treatment from thesame patient. A long catheter needle was placed in the center of thislarge liver cancer and 20 mL of the “medicinal ion bomb” solution wasinjected. FIG. 15.3. The cancer became smaller from the pretreatment 67mm×67 mm×80 mm to posttreatment 45 mm×45 mm×60 mm in size 7 days aftertreatment.

Example 16

FIG. 16 shows the case of a lady who suffered from breast fibrocysticchange in the inventor's clinical study group. FIG. 16.1. An image ofpretreatment mammography shows a large cyst at 40 mm×40 mm in size thatis located behind of the left nipple and several small fibrocysticlesions are surrounding the large cyst. FIG. 16.2. The mucosa-like fluidinside the cyst was sucked, and the large cyst is repeatedly washed andsucked. Then a single intra-cystic injection with 3.5 mL of the“medicinal ion bomb” was performed. FIG. 16.3. The image of theposttreatment mammography to demonstrate the fibrocystic breast diseasewas cured 3 months after treatment.

Example 17

FIG. 17 shows the case of a 78 year-old man who suffered from lipoma.This tumor was located in his rear neck for 18 years. FIG. 17A. Thepretreatment tumor was 40 mm×40 mm in size. FIG. 17B. A singleintratumoral injection with 2.0 mL of the “medicinal ion bomb” wasadministered into the tumor. FIG. 17C. The large tumor was cured by 8weeks. A 9 year follow-up study showed no recurrence of the treatedtumor.

Example 18

FIG. 18 shows an example where a patient who suffered from atheroma.FIG. 18A. A pretreatment atheroma in the vertex was 18 mm×18 mm in size.FIG. 18B. A counterpart photo of the video image to demonstrate a singleintratumoral injection with 0.5 mL of the “medicinal ion bomb.” FIG.18C. The atheroma was cured in 2001. This photo was taken when a 9 yearfollow-up study was performed.

Example 19

FIG. 19 shows the case of a 66 year-old man who suffered from a largeatheroma at 22 mm×28 mm in size. The tumor was treated using a singleintratumoral injection with 1.5 mL of the “medicinal ion bomb” in 2001.A 9 year follow-up study showed no recurrence of the treated tumor.

Example 20

FIG. 20 shows the application of intralesional injection with the“medicinal ion bomb” in cosmetic conditions. FIG. 20.1 shows a skin molein the patient's face. FIG. 20.2. A single intralesional injection with0.2 mL of the “medicinal ion bomb”. FIG. 20.3. The mole has developedcoagulative necrosis by 7 days posttreatment. FIG. 20.4. The skin molewas removed without scar defect.

Supplemental Examples and Materials Preparations of the “Medicinal IonBomb” and the “Medicinal Ion Bomb” Solution

The “medicinal ion bomb solution” here refers to the composition asshown in Table 1.

TABLE 1 Preparation of the “Medicinal Ion Bomb” Solution 2 MainComponents Water Added (mL) Final Volume (mL) 295 g NaCl 890 1,000 27.75g CaCl₂ 29.5 g NaCl 89 100 2.775 g CaCl₂ 14.75 g NaCl 44.5 50 1.3875 gCaCl₂ 7.375 g NaCl 22.25 25 0.694 g CaCl₂

Solubility of the “Medicinal Ion Bomb”

The compositions of the “medicinal ion bomb” comprise 5.0479 M of NaCland 250 mM of CaCl₂ that are completely dissolved in about 890 mL ofdistilled water at room temperature by 7 minutes, making the finalvolume of one liter of the “medicinal ion bomb” solution (the saturatedionic solution).

pH Value of the “Medicinal Ion Bomb” Solution

The pH value of the “medicinal ion bomb” solution is 7.32±at roomtemperature.

The “Medicinal Ion Bomb” and the “Medicinal Ion Bomb” Solution

The “medicinal ion bomb” for cancer treatment in this study project canbe made in two forms. One is in the form of powders of NaCl and CaCl₂that is called the “medicinal ion bomb”; and the other one is in theform of liquid that is termed the “medicinal ion bomb” solution.Generally, the terms of the “medicinal ion bomb” and the “medicinal ionbomb” solution in this study project can be interchangeable.

Intratumoral Injection and Precision-Guided Intratumoral Injection withthe “Medicinal Ion Bomb”

When the “medicinal ion bomb” solution is used to treat a tumor orcancer in the surface of the body, skin, or subcutaneous tissue, thetreatment is performed under direct view that is termed intratumoralinjection with the “medicinal ion bomb.” When a tumor or cancer islocated in deep tissue or internal organ, the treatment is accuratelyperformed by means of CT scan guidance or ultrasound guidance that istermed the precision-guided intratumoral injection with the “medicinalion bomb.”

In Vivo Toxicity of Na⁺, K⁺, Ca²⁺ and Mg²⁺

At the beginning, we examined in vivo toxicity of high concentrations ofNa⁺, Ca²⁺, K⁺, and Mg²⁺ in BALB/c mice. Here, the models of theintramuscular injection using 35% NaCl, 35% CaCl₂, 34% KCl and 35% MgCl₂(each solution contains 20 mg of adrenaline/liter as its dose in the“medicinal ion bomb” solution) represent the model of intratumoralinjection with the above 4 types of ionic solutions.

BALB/c mice were housed and inbreeded in the contracted universityanimal facility that is satisfied of the regulation of NIH criterion.Body weight of the BALB/c mice at the experiment was 30±grams. Thetoxicity test was performed using intramuscular injection with 0.2 ml of35% NaCl, 35% CaCl₂, 34% KCl or 35% MgCl₂ in a mouse, respectively.

In total, 20 BALB/c mice (10 males and 10 females) were randomly dividedinto 5 groups, with 4 mice in each group.

Animals in group 1 received intramuscular injection with 0.2 mL of 35%NaCl and survived the experiment, indicating that the dose ofintramuscular injection with 0.2 ml of 35% NaCl solution is safe inmouse weighing 30 grams.

Animals in group 2 received intramuscular injection with 0.2 mL of 35%CaCl₂ and died in 3 hours after treatment, indicating that highconcentration of CaCl₂ is toxic.

Animals in group 3 received intramuscular injection with 0.2 ml of 17.5%CaCl₂ and survived the experiment, indicating that intramuscularinjection with low dose of CaCl₂ is safe.

Animals in group 4 received intramuscular injection with 0.2 mL of 34%KCl and died from cardiopulmonary failure 3 hours after treatment,indicating that high concentration of KCL is toxic, that cannot be usedto be an component in the formula of the “medicinal ion bomb”.

Animals in group 5 received intramuscular injection with 0.2 mL of 35%MgCl₂ and died in 3 hours after treatment, indicating that highconcentration of MgCl₂ is toxic.

Ex Vivo Killing Action of the “Medicinal Ion Bomb” to 18 Types of HumanCancer Cell Lines

The role of the “medicinal ion bomb” in killing cancer cells was testedin tissue culture. Tumor cells were seeded in Petri dish using DMEM orRPMI1640 medium supplemented with 10% FCS. Each type of tumor cell linewas prepared at 2×10⁶ cells/mL in a 5 mL plastic tube. The supernant wasremoved by centrifugation and then 1.0 mL of the “medicinal ion bomb”solution was added to each tube. Three to five minutes later, 0.5%Trypan Blue was added and mixed with the cell suspension for 2 minutes.The number of dead cancer cells was counted under an invertedmicroscope. Selected samples of treated cancer cell suspension for flowcytometry were prepared in phosphate-buffered saline (PBS) and mortalityof cells was assayed using flow cytometer (Beckman Coulter Inc., CA).

This experiment included one control group in which cancer cells weretreated with normal saline, and the other 18 types of human cancer celllines were treated with the “medicinal ion bomb” solution. The datashowed that no cancer cells were dead in the control group. However, all18 types of human cancer cell lines were killed by the “medicinal ionbomb” solution in 3 to 30 minutes, including A549 human lung cancer, AROhuman thyroid cancer, CWR-22 human prostate cancer, HeLa human cervicalcancer, HL60 human leukemia, HT-29 human colorectal cancer, Jurkat humanT cell leukemia, K562 human leukemia, M2 human lymphoma, M24 Humanmelanoma, MCF7 human breast cancer, MDA-MB-231 human breastadenocarcinoma, OVCAR3 human ovary cancer, PC3 human prostate cancer,SK-RC-52 human kidney cancer, T-47D human breast epithelial tumor, LNCaPhuman prostate cancer, and U87 human glioblastoma.

Intratumoral Injection LD50 of the “Medicinal Ion Bomb” Solution inMouse and Human

In toxicology, the median lethal dose (LD₅₀) of a toxin, radiation, orpathogen is the dose required to kill half the members of a testedpopulation after a specified test duration. LD₅₀ figures are frequentlyused as a general indicator of a substance's acute toxicity.

Before conducting the experimentation of the LD₅₀ in this study, overhundreds of nude mice bearing tumor underwent intratumoral injectionwith the “medicinal ion bomb.” The experience from experimentaltreatment showed that the LD₅₀ of the “medicinal ion bomb” forintratumoral injection is 0.15±ml in a nude mouse weighing 20±grams.

In this group, 12 nude mice bearing MCF7 human breast cancer weretested. Each nude mouse was subcutaneously inoculated with a MCF7 humanbreast cancer. The experiment started, when tumors reached 8 mm×8 mm insize, with an injection of 0.15 mL of the “medicinal ion bomb” solutionto the tumor. The time and number of animal death and survival, tumorsize, and samples for pathology were collected for statistical analysis.

NaCl and CaCl₂ are not generally considered poisonous. Our data haveconfirmed that the intratumoral injection LD₅₀ of the “medicinal ionbomb” is 7.5 mL/kilogram (kg) in mouse. According to Pharmacology andToxicology, Guidance for Industry: Estimating the maximum safe startingdose in initial clinical trials for therapeutics in adult healthyvolunteers. FDA, July 2005, because the conversion factor of mouse drugdose to human equivalent dose is 0.081, the intratumoral injection LD₅₀of the “medicinal ion bomb” solution in human is calculated by:

7.5  mL/kg  mouse  drug  dose × 0.081 = 0.6075  mL/kg  human  equivalent  dose  (HED) = 179.213  mg/kg  NaCl + 16.86  mg/kg  CaCl₂(HED)

The data of the invention confirmed that the intratumoral injection LD₅₀of the “medicinal ion bomb” solution” in nude mouse weighing 20±grams is0.15 mL. Shown below are the calculation steps of intratumoral injectionLD₅₀ of the “medicinal ion bomb” solution from mouse drug dose to HED.

Intratumoral Injection LD50 of the “Medicinal Ion Bomb” Solution inMouse is Calculated by

Average body weight of nude mice ≈20 grams.

1.0 kg=1,000 grams.

1,000 grams/20 grams=50 folds.

Intratumoral injection LD₅₀ of the “medicinal ion bomb” solution in anude mouse is 0.15 mL.

0.15 mL×50 folds=7.5 mL/kg “medicinal ion bomb” solution in mouse.

7.5 mL/kg×295=2212.5 mg/kg NaCl in mouse LD₅₀.

Here the 295 means 295 grams of NaCl in one liter of the “medicinal ionbomb” solution.

7.5 mL/kg×27.75=208.125 mg/kg CaCl₂ in mouse LD₅₀.

Here the 27.75 means 27.75 grams of CaCl₂ in one liter of the “medicinalion bomb” solution.

Intratumoral injection LD₅₀ of the “medicinal ion bomb” solution in nudemouse

-   -   is 7.5 mL/kg body weight or    -   =2212.5 mg/kg NaCl+208.125 mg/kg CaCl₂.

Intratumoral Injection LD50 of the “Medicinal Ion Bomb” Solution inHuman is Calculated by

Intratumoral injection LD₅₀ of the “medicinal ion bomb” solution inmouse is 7.5 mL/kg body weight.

Here 0.081 is the conversion factor of mouse drug dose in mg/kg to HEDin mg/kg¹. ¹ Guidance for Industry. Estimating the maximum safe startingdose in initial clinical trials for therapeutics in adult healthyvolunteers. Pharmacology and Toxicology, FDA, July 2005.

7.5 mL×0.081=0.6075 mL/kg “medicinal ion bomb” solution in HED.

0.6075 mL/kg×295=179.213 mg/kg NaCl in HED.

Here the 295 means 295 grams of NaCl in in one liter of the “medicinalion bomb” solution.

0.6075 mL/kg×27.75=16.86 mg/kg CaCl₂ in HED.

Here the 27.75 means 27.75 grams of CaCl₂ in one liter of the “medicinalion bomb” solution.

Intratumoral injection LD₅₀ of the “medicinal ion bomb” solution inhuman

-   -   is 0.6075 mL/kg body weight or    -   =179.213 mg/kg NaCl+16.86 mg/kg CaCl₂.

Intratumoral Injection MTD of the “Medicinal Ion Bomb” Solution in Mouseand Human

Maximum tolerated dose (MTD) refers to the highest dose of aradiological or pharmacological treatment that will produce the desiredeffect without unacceptable toxicity.

In this group, 15 nude mice bearing MCF7 human breast cancer weredivided into 5 groups with 3 animals in each subgroup. Animals insubgroup-1 received intratumoral injection with 0.15 mL of the normalsaline as the control. Animals in subgroup-2 received intratumoralinjection with 0.12 mL of the “medicinal ion bomb” solution. Animals insubgroup-3 received intratumoral injection with 0.13 mL of the“medicinal ion bomb” solution. Animals in subgroup-4 receivedintratumoral injection with 0.14 mL of the “medicinal ion bomb”solution. And animals in subgroup-5 received intratumoral injection with0.15 mL of the “medicinal ion bomb” solution. Each nude mouse wassubcutaneously inoculated with a MCF7 human breast cancer. Theexperiment started, upon tumor growth at 8 mm×8 mm in size. Theexperimental data confirmed that the intratumoral injection MTD of the“medicinal ion bomb” solution in nude mouse weighing 20 grams is 0.14mL. Shown below are the calculation steps of the intratumoral injectionMTD of the “medicinal ion bomb” solution from mouse to human.

Average body weight of nude mice ≈20 grams.

1.0 kg=1,000 grams.

1,000 grams/20 grams=50 folds.

Intratumoral injection MTD of the “medicinal ion bomb” solution is 0.14mL in a nude mouse.

0.14 mL×50 folds=7.0 mL/kg of the “medicinal ion bomb” solution inmouse.

Here 0.081 is the conversion factor of mouse drug dose in mg/kg to HEDin mg/kg.

7.0 mL×0.081=0.567 mL/kg “medicinal ion bomb” solution in human.

0.567 mL/kg×295=167.265 mg/kg NaCl in human.

Here the 295 means 295 grams of NaCl in one liter of the “medicinal ionbomb” solution.

0.567 mL/kg×27.75=15.734 mg/kg CaCl₂ in human.

Here the 27.75 means 27.75 grams of CaCl₂ in one liter of the “medicinalion bomb” solution.

The intratumoral injection MTD of the “medicinal ion bomb” solution is7.0 mL/kg in mouse, the conversion factor of mouse drug dose to HED is0.081. Here the intratumoral injection MTD of the “medicinal ion bomb”in human is calculated by:

7.0 mL/kg mouse drug dose×0.081=0.567 mL/kg in HED=167.265 mg/kgNaCl+15.734 mg/kg CaCl₂ in HED

Tumor Size, Tumor Weight, Blood Volume and Volume of “Medicinal IonBomb” Solution

As described previously, we conducted ex vivo experiments of thediffusion of the “medicinal ion bomb” solution in beef liver, in vivoexperiments of the diffusion of the “medicinal ion bomb” in the liver ofalive rat and rabbit, and clinical study in 78 human patients whosuffered from various tumors. According to human physiology, bloodvolume is about 80 mL/kg body weight in adult. The data showed that atumor at 2.5 cm×2.5 cm×3.0 cm in human patient was killed by about 1.0mL of the “medicinal ion bomb” solution. A solid tumor was at 4.5 cm×6cm in size in human patient that was killed by approximately 5 to 6 mLof the “medicinal ion bomb” solution. Further, a solid tumor at 7 cm×8cm in size that was killed by about 20 mL of the “medicinal ion bomb”solution. The estimated volume of the “medicinal ion bomb” in differentsizes of tumor is calculated in Table 2 below.

TABLE 2 Calculation of the Required Volume of the “Medicinal Ion Bomb”in Different Sizes of Tumor Tumor Blood Volume Ion Bomb Solution TumorSize Weight (g) (80 mL/kg) Needed (mL)  1 cm × 1 cm × 1 cm 1.25 0.120.12-0.15  2 cm × 2 cm × 2 cm 8.6 0.69 0.5-1.0  3 cm × 3 cm × 3 cm 282.24 1.0-2.0  4 cm × 4 cm × 4 cm 55 4.4 2.0-3.0  5 cm × 5 cm × 5 cm 16213 5.0-10   6 cm × 6 cm × 6 cm 255 20.4 10-20  7 cm × 7 cm × 7 cm 42033.6 30-40  8 cm × 8 cm × 8 cm 590 47.2 40-50  9 cm × 9 cm × 9 cm 72654.1 50-60 10 cm × 10 cm × 10 cm 1,150 80 60-80

Table 2 shows three repeated measurements of ex vivo tumor size andtumor weight that were examined using fresh en bloc pork meats. Thecalculation of blood volume in tumor tissue is cited from the OxfordFood & Nutrition Dictionary in which the average blood volume is 5.3 L(78 mL/kg body weight) in male and 3.8 L (56 mL/kg body weight) infemale. Here, the constant “80 mL” of blood volume per kilogram bodyweight is used to calculate blood volume in different sizes of tumor.

FIG. S1. This group of pictures is the description of clinical data ofthe invention that is useful for clinical investigator, physician andoncologist to estimate the required amount of the “medicinal ion bomb”solution in different sizes of tumor. FIG. S1.1 is a picture of a largegrape at 2.5 cm×2.5 cm×3.0 cm in size (weighing 10.1 grams). Clinically,such size of a cancer or benign tumor is killed by one singleintratumoral injection with 1.0 mL of the “medicinal ion bomb” solution.FIG. S1.2 is a picture of an egg at 4.5 cm×4.5 cm×6.0 cm in size(weighing 69 grams), requiring approximately 6.0 mL of the “medicinalion bomb” solution for the treatment. FIG. S1.3 is a picture of a freshGala apple at 7.0 cm×7.0 cm×8.0 cm in size (weighing 290 grams), whichneeds about 20 mL of the “medicinal ion bomb” solution.

Tissue Damage in the Injection Site of the “Medicinal Ion Bomb” Solution

Intramuscular injection with the “medicinal ion bomb” solution onlycaused mild injury to normal muscular tissue in the injection site.Microscopically, there was unremarkable damage to muscular tissue exceptmomentary infiltration of inflammatory cells. For detailed description,see FIG. S5.

Acute Toxicity Test and Chronic Toxicity Test of the “Medicinal IonBomb”

In this study, acute toxicity test, including liver function, kidneyfunction, pancreatic and pulmonary functions, and blood electrolytes(Na⁺, K⁺ and Ca²⁺), were examined in rats.

Thirty-six normal Sprague Daley (SD) rats weighing 220±grams weredivided into 5 groups with 4 animals in the control group and 8 rats ineach experimental group. Each animal in the control group received 1.0mL of normal saline through intramuscular injection (the same amount ofthe “medicinal ion bomb” solution as in the experimental groups).Animals in the experimental groups 2, 3, 4 and 5 were treated throughintramuscular injection with 1.0 mL of the “medicinal ion bomb” solution(the MTD “medicinal ion bomb” solution containing 20 mg/mL adrenaline)in rats.

Animals in Group 1 were the controls and they were killed for artery(abdominal aorta) blood sample on day zero. For acute toxicity test,animals in Group 2 were sampled on day one; animals in Group 3 weresampled on day two; and animals in Group 4 were sampled on day threeafter treatment. For chronic toxicity test, animals in Group 5 weresampled on day 66 after treatment. The laboratory parameters include:

Liver function (blood ALT, total bilirubin, and total protein)

Kidney function (blood urea nitrogen, BUN)

Pancreatic function (blood sugar reflects partially pancreatic function)

Lung function (partial pressure CO₂, PCO₂) and

Electrolytes (K⁺, Na⁺ and Ca²⁺).

Samples were tested using a Hitachi Automatic Biochemical Analyzer 717(Roche Diagnostics).

Data from the control group and experimental groups were expressed asmean values±SD. Results between the normal control group andexperimental groups were compared by analysis of variance and Student'st-test using EXCEL statistical software. A p value <0.05 was consideredstatistically significant, with p<0.01 being highly significant.

Liver

The results showed that when the MTD “medicinal ion bomb” solution wasintramuscularly administered, the “medicinal ion bomb” solution causedtransiently mild injury to the liver function that was recovered in 4days after treatment. For the detailed data, see FIG. S2. FIG. S2.1. Foracute toxicity test of liver function, the peak level of blood ALT isseen in one and two days after treatment, and it returns to normal rangein 4 days after treatment. FIG. S2.2. Increased level of blood totalbilirubin (it is still in normal range) is seen in the first 2 days andreturns to normal in 4 days after treatment. FIG. S2.3. Blood totalprotein decreases in the first 3 days and returns to normal by 4 daysafter treatment. These parameters indicate that increased levels ofblood ALT and bilirubin, and decreased level of blood total protein aretransitory. Sixty-six days after treatment, levels of blood ALT,bilirubin, and total protein are normal, indicating that the “medicinalion bomb” has no chronic toxicity to liver function.

As seen in FIG. S2 there is no evidence of chronic toxicity caused bythe “medicinal ion bomb” solution to liver function.

Kidney

The level of BUN (normal range 10-20 mg/dl) increased from the controllevel of 7.73 to 11.82 mg/dl (it was still in normal range) in one dayafter treatment. BUN dramatically declined to 8.0 mg/dl in two days andreturned to the control level in 4 days after treatment. As seen in FIG.S3.1, there is no evidence that the “medicinal ion bomb” solution hascaused chronic toxicity to the kidney.

Pancreas

The level of blood sugar can partially reflect the function of thepancreas. One and 2 days after treatment, the level of blood sugar waslower than the control level. Blood sugar returned to the control levelby 4 to 5 days after treatment. As shown in FIG. S3.2 there is noevidence of chronic toxicity of the “medicinal ion bomb” to thepancreas.

Lung

PCO₂ was only tested on day one after treatment. As seen in FIG. S3.3,there was no significant difference in PCO₂ between the control leveland posttreatment one, indicating that there was no acute toxicity ofthe MTD “medicinal ion bomb” treatment to lung function. FIG. S3.3. Theyellow column represents PCO₂ in the control group and blue columnrepresents PCO₂ in the experimental group treated with the MTD“medicinal ion bomb” solution. Making comparison of PCO₂ levels betweenthe experimental group and control, P>0.05 and there is no statisticaldifference.

Heart

The affect of the “medicinal ion bomb” on heart rate, blood pressure andbreathing was examined in SD rats during and after treatment using acomputer-assisted Buxco Max II Biosystem (Buxco Electronics, Sharon,Conn.). No abnormal findings were found in the above 3 parameters.

It must point out that the results of these parameters are resulted fromthe MTD “medicinal ion bomb.” This is not an issue in clinical practice.Generally, the dimension of a treated human tumor is smaller than 10cm×10 cm in diameters that needs the amount of the “medicinal ion bomb”is much less than the MTD. Therefore, the mild and transitoryside-effects of the liver function from the MTD “medicinal ion bomb” canbe ignored.

Pharmacokinetics of the “Ion Bomb Solution”

Pharmacokinetic models predict the time dependence of a drug'sconcentration in the body fluids following its administration.Pharmacokinetics is mainly divided into four areas including the extentand rate of absorption, distribution, metabolism and excretion (ADME).In this study, the pharmacokinetics of intratumoral injection with the“medicinal ion bomb solution” was studied in rats.

Sixty SD rats, both genders, weighing 200±grams, were randomly dividedinto 30 subgroups with 2 animals in each subgroup. Animals in thecontrol group received 0.5 mL of normal saline intramuscularly. Allanimals in experimental groups received intramuscular injection with 0.5mL of the “medicinal ion bomb” solution (containing 20 μg/mL adrenaline)in the buttock (the MTD of the “medicinal ion bomb” solution in ratweighing 200±grams is 1.0 mL. In this experiment, the ½ amount of theMTD “medicinal ion bomb solution” was used). Blood samples were obtainedthrough the abdominal aorta at an interval of every 30 minutes,consecutively from the start time point 0 to the end time point of 15hours after treatment. The concentrations of plasma Na⁺ and Ca²⁺ inblood samples were analyzed using a Hitachi Automatic BiochemicalAnalyzer 717. Statistically, mean values of the control group and allexperimental groups were used to plot the pharmacokinetic patterns ofplasma Na⁺ and Ca²⁺.

As shown in FIG. S4, the ascending concentrations of plasma Na⁺ to 50%,63% and 95% were found at posttreatment points of 2.0, 2.5 and 3.0hours, respectively. The peak level of plasma Na⁺ was detected at 3.5hours after treatment. The levels of plasma Na⁺ were decayed to 50%, 37%and 5% at 7.0, 8.0 and 11 hours after treatment, respectively.

The ascending concentrations of plasma Ca²⁺ to 50%, 63% and 95% were atposttreatment points of 2.0, 2.5 and 3.0 hours, respectively. It decayedto 50%, 37% and 5% at posttreatment points of 7.0, 8.0 and 11 hours,respectively.

The data showed that 95% clearance of the injected Na⁺ and Ca²⁺ fromblood was seen at 11 hours after intramuscular injection with the“medicinal ion bomb.” Most of NaCl and CaCl₂ are excreted through thekidney and gastrointestinal tract. Both concentrations of plasma Na⁺ andCa²⁺ returned to the control levels at 15 hours after intramuscularinjection.

FIGS. S4.1 and S4.2 show the time points of the ascending concentrationsof plasma Na⁺ and Ca²⁺ to 50%, 63% and 95% and the time points of theelimination concentrations of plasma Na⁺ and Ca²⁺ to 50%, 37% and 5% inthe model of intratumoral injection with the “medicinal ion bomb”solution in SD rat.

Half-Life of Plasma Na⁺ and Ca²⁺ from the “Medicinal Ion Bomb” Solution

A biological half-life or elimination half-life is the time that ittakes for a drug to lose one-half of its pharmacologic activity. Asshown in FIG. S4, the half-life of plasma Na⁺ and Ca²⁺ from the“medicinal ion bomb” solution is detected at 7.0 hours afterintratumoral injection in SD rat.

Pharmacodynamic Study on the “Medicinal Ion Bomb Solution”

Pharmacodynamic models deal with the action of the drug once it reachesits target organ. Pharmacodynamic actions of the “medicinal ion bomb”solution to kill cancer cells are multiple-directions. However, as shownby the data of the patch clamp technique, fluorescent Na⁺ and Ca²⁺channel probe monitoring, the real-time microscopic video, pathology andSEM and TEM, it mainly targets the cell membranes and the compositionsof the nucleus, mitochondria, lysosome and other organelles of cancercells as well as tumor vessels.

The cell membrane is a biological membrane that separates the interiorof all cells from the outside environment. The cell membrane isselectively permeable to ions and organic molecules, and controls themovement of substances in and out of cells. The basic function of thecell membrane is to protect the cell from its surroundings. If themembrane of a cell is damaged or broken, the fate of the cell is death.

Membrane potential is the difference in electric potential between theinterior and exterior of a cell. Membrane potential of cells can bemeasured by the patch clamp technique that allows the study of single ormultiple ion channels in cells, especially it is used in the study ofexcitable cells such as neurons, cardiomyocytes, muscle fibers andpancreatic beta cells. Changes in membrane potential can also be used toevaluate the death of cardiomyocyte in vitro.

Previously, we introduced the application of the patch clamp techniquein determination of membrane potential of mouse cardiomyocyte. When alow concentration of the “medicinal ion bomb” solution was administered,death of treated cells is characterized by a sub-acute pathologicprocess. For example, the mouse cardiomyocyte was killed in 5 minutesafter treatment with 5% concentration (252.4 mM of Na⁺ and 12.5 mM ofCa²⁺) of the “medicinal ion bomb” solution. The mouse cardiomyocyte wasdead in 3 minutes after treatment with 10% concentration (504.79 mM ofNa⁺ and 25 mM of Ca²⁺) of the “medicinal ion bomb solution”. When the100% “medicinal ion bomb” solution (5.0479 M of Na⁺ and 250 mM of Ca²⁺)was used, death of treated cancer cells is characterized by an acutepathologic process. Under the real-time inverted microscope, MCF7 humanbreast cancer cells in tissue culture were killed in 12 to 18 seconds inthe standard “medicinal ion bomb” solution. This fact indicates thatpharmacodynamic action of the “medicinal ion bomb” solution to killcancer cells is the dose- and time-dependence.

Histopathologic Study on Vital Organs

Histopathologic study was performed on 6 vital organs in mice treatedwith the “medicinal ion bomb.” Specimens were taken 24 hours aftertreatment with the “medicinal ion bomb” solution and fixed in formalin,embedded in paraffin, stained with hematoxylin and eosin, and read undera digital light microscope by two experienced pathologists. The datadisclosed that the treated animals didn't show evidence of pathologicdamage to heart, lung, liver, kidney, pancreas and uterus (FIG. S5).

FIG. S5.1 is a muscular tissue from the injection site of the “medicinalion bomb” solution in mouse. The muscular tissue shows mild injury withinfiltration of inflammatory cells. FIG. S5.2. Normal microscopicfeature of the heart. FIG. S5.3. Normal lung. Magnifications ×200.

FIGS. S5.4-S5.6 show normal microscopic features of the liver, pancreasand kidney. Magnifications ×200.

Theory and Mechanism of the “Medicinal Ion Bomb” for Treating Cancer,Tumor and Neoplasm

Regarding the theory and mechanism of the “medicinal ion bomb” forcancer treatment, as shown in FIGS. S6 and S7, they are related tobiochemistry, cell biology, cell physiology, cell membrane, cellularmembrane potential, ion channels, ion tracking techniques, and theformula of the “medicinal ion bomb” solution using high concentrationsof Na⁺ and Ca²⁺ between the extracellular and intracellular fluids.

A chance opportunity sparked this invention. One day, the inventor wentshopping in a Chinese supermarket and bought a dozen salted duck eggs.On the way back home, he wondered why these salted duck eggs can bepreserved at room temperature for years without rotting. The answer isthat proteins and yolks in salted duck eggs are coagulated or clotted byhigh concentration of sodium chloride, in particular by highconcentration of Na⁺. The inventor presumed that high concentration ofNa⁺ might coagulate or clot proteins in cancer cells like what occurs insalted duck eggs.

In cell physiology, survival of normal cells, tumor cells or cancercells is dependent upon the equilibrium of osmotic pressure inside andoutside of cells. Normal osmotic pressure of cells depends on theequilibrium of concentrations of ions between the extracellular andintracellular fluids. When concentrations of ions between theextracellular and intracellular fluids are identical, it is calledisotonic. Since the cell is at equilibrium, there is no ionicconcentration gradient, and the flow of water in is equal to the flow ofwater out. This does not cause sickness or death of the cell.

In physiological condition, the concentration of Na⁺ in theextracellular fluid is 140 mM and intracellular fluid is 14 mM. Becausethe “medicinal ion bomb” solution has 5.0479 M of Na⁺ the concentrationof Na⁺ in the extracellular fluid of cancer cells is 36-fold higher thanthe normal level, and the concentration of Na⁺ in the intracellularfluid is about 360-fold higher than the normal concentration. When alarge amount of Na⁺ crosses the cell membrane from the extracellularfluid to intracellular fluid by osmosis, cancer cells are killed indozen seconds by overwhelming influx of high concentration of Na⁺ fromthe “medicinal ion bomb” (FIGS. S6 and S7). This theory has been provenby the patch clamp technique for membrane potential and fluorescent ionchannel probe monitor system.

FIG. S6.1 is a salted duck egg which was prepared with sodium chloride 2years ago. FIG. S6.2 shows a salted duck egg which has been cooked andcut. Its albumin and yolk have not rotted even though it was preservedat room temperature for 2 years. FIG. S6.3 shows a picture of a cancercell and the “medicinal ion bomb” comprising 5.0479 M of Na⁺ and 250 mMof Ca²⁺ in the extracellular fluid.

On the other hand, the normal concentration of Ca²⁺ in the extracellularfluid is 2.5 mM and intracellular fluid is 0.0001 mM in mammalian cells.The “medicinal ion bomb” contains 250 mM of Ca²⁺ which is 100-foldhigher than its physiological level in the extracellular fluid andapproximately 2.5 million-fold higher than its physiologicalconcentration in the intracellular fluid. As a result, when a highconcentration of Ca²⁺ overwhelmingly crosses the cell membrane from theextracellular fluid to intracellular fluid, cancer cells are killedimmediately. This theory has also been confirmed by the patch clamptechnique and ionic tracking techniques in the invention (FIGS. S8-S10).

FIG. S7 shows a fragment of the cell membrane where normal levels of Na⁺and Ca²⁺ in the extracellular fluid and high concentrations of Na⁺ andCa²⁺ in the “medicinal ion bomb” are seen. Channels of Na⁺, Ca²⁺, K⁺ andothers are embedded in the cell membrane. When extremely high levels ofNa⁺ and Ca²⁺ influx from the extracellular fluid to intracellular fluid,cancer cells are killed in dozen seconds.

More importantly, Ca²⁺ is one of secondary messengers in cell signaltransduction biology. High concentration of Ca²⁺ in the intracellularfluid not only causes tremendous physiological damages, but also causeshuge biological actions to cancer cells. Therefore, high concentrationof Ca²⁺ in the “medicinal ion bomb” solution can accelerate death ofcancer cells and it is regarded as the enhancer of cancer-killing.

FIG. S8. Three diagrams of the patch clamp technique in testing of themembrane potential of mouse cardiomyocytes treated with differentconcentrations of the “medicinal ion bomb” solution. FIG. S8.1 is thecontrol membrane potential of a normal mouse cardiomyocyte in TyRodesolution. FIG. S8.2 shows that a mouse cardiomyocyte is dead in 5minutes after treatment with 5% concentration of the “medicinal ionbomb” solution. FIG. S8.3. A mouse cardiomyocyte is dead in 3 minutesafter treatment with 10% concentration of the “medicinal ion bomb”solution. The data show that the killing action of the “medicinal ionbomb” solution to tumor cells is dose- and time-dependence.

FIG. S9 is a group of dynamic pictures of Na⁺ influx using fluorescentNa⁺ channel probe system. FIG. S9.1 shows the gated 6 pretreatment MCF7human breast cancer cells. FIG. 59.2 demonstrates that 2 of them aredead by 10 seconds after starting the treatment with the “medicinal ionbomb” solution. FIG. S9.3 depicts that all gated cancer cells are deadby 16 seconds after starting the treatment with the “medicinal ion bomb”solution. These pictures are the counterparts from the monitor of aMacintosh computer.

FIG. S10 is a group of dynamic pictures of Ca²⁺ influx using fluorescentCa²⁺ channel probe system in MCF7 human breast cancer cells. FIG. S10.1shows the gated 7 cancer cells before the treatment. FIG. S10.2demonstrates that 2 of 7 cancer cells are dead by 9 seconds afterstarting the treatment with the “medicinal ion bomb” solution. FIG.S10.3. Four of 7 cancer cells are dead by 12 seconds after starting thetreatment. FIG. S10.4 depicts that 5 of 7 cancer cells are dead by 16seconds after starting the treatment. FIG. S10.5. All 7 cancer cells arekilled by 18±seconds after starting the treatment. These pictures arethe counterparts from the monitor of a Macintosh computer.

Enhance Cancer-Killing Actions of the Combination of Na⁺ and Ca²⁺

In this invention, we studied the influx of Na⁺ and Ca²⁺ from theextracellular fluid into intracellular fluid of cancer cells. Thefluorescent Na⁺ or Ca²⁺ probe monitoring showed that use of 5.0479 M ofNa⁺ solution alone took 20±4 seconds to kill MCF7 human breast cancercells. When the “medicinal ion bomb” solution was used, it only took18±4 seconds to kill the same type of cancer cells, which indicates thatCa²⁺ has an enhancer effect in cancer treatment and the formula ofcombination of Na⁺ and Ca²⁺ has synergetic and complementarycancer-killing effect.

We observed that the diffusion of Na⁺ in tumor tissue in living animalsis fast and the diffusion of Ca²⁺ is slow, and both types of ions inhigh concentration are capable of killing cancers when they are usedseparately. We have confirmed that the combination of Na⁺ and Ca²⁺ inthe formula of the “medicinal ion bomb” has synergetic, complementaryand dual-wave cancer-killing effects. The first wave of cancer-killingeffects (the early killing action) is mediated by Na⁺, which happens inone to 12 hours after treatment. The second wave of cancer-killingaction (the later killing effect) is mediated by Ca²⁺, which occurs in 6to 24 hours after treatment.

Five Phases of the “Medicinal Ion Bomb” to Kill Cancer Cells

The video data of real-time inverted microscopy revealed 5 pathologicphases of the “medicinal ion bomb” to kill cancer cells: (1) Rapiddisruption of the membrane of cancer cells; (2) acute intracellulardehydration of cancer cells because of high concentrations of Na⁺ andCa²⁺ in the extracellular fluid after adding the “medicinal ion bomb”into the cell culture chamber; (3) swelling of cancer cells due to theinflux of large amounts of Na⁺ and Ca²⁺ from the extracellular fluidinto intracellular fluid; (4) bursting of cancer cells caused by extremeswelling; and (5) consequential death of cancer cells. The entireprocess of 5 pathologic phase damages to cancer cells occurred in dozenseconds and each of the pathologic phases occurred in only 3 to 4seconds (FIGS. S11-S13).

Pathology Findings

Macroscopically, pathologic features of intratumoral injection with the“medicinal ion bomb” solution in human patients, as shown in thepictures below, the treated tumors undergo 4 pathologic features. (1)From 1 to 12 hours after treatment, tumor cells or cancer tissueexperience in situ degeneration and necrosis and destruction of tumorvessels. The surface of the treated tumor appears light blue. (2) If atumor or cancer is smaller than 10 mm×10 mm in size, it will becompletely removed by one intratumoral injection with the “medicinal ionbomb” in 24 to 48 hours. When a tumor or cancer is larger than 20 mm×20mm in size, the treated tumor develops coagulative necrosis with acharred, dark, and dead tumor remainder. (3) The dead tumor or cancerremainder comes off leaving a small wound in 7 to 10 days aftertreatment. (4) The wound of the treated tumor is generally healed by 2to 3 weeks posttreatment.

Histopathologic findings of human cancer treated with the “medicinal ionbomb” were featured: (I) Na⁺ and Ca²⁺ salts infiltrated in thesurrounding tissue of cancer cells (FIG. S14); (II) necrosis of tumortissue and cancer cells is seen where the “medicinal ion bomb” solutionreached, indicating that fully filling a tumor with the “medicinal ionbomb” solution is a necessary factor to kill a primary cancer; (III)thromboses in tumor blood vessels; and (IV) massive bleeding in necroticareas resulting from injured tumor blood vasculatures. For detaileddata, see FIGS. S15 and S16.

FIG. S11. This is a group of dynamic pictures of real-time invertedmicroscopy on MCF7 human breast cancer cells. FIG. S11.1 depicts thepretreatment cancer cells as the control. FIG. S11.2 shows slightlychanges in cell morphology 3 seconds after starting the treatment withthe “medicinal ion bomb.” FIG. S11.3 is 5 seconds after starting thetreatment. All treated cancer cells are heavily damaged. FIG. S11.4 is16 seconds after starting the treatment. All treated cancer cells inthis picture are dead and smashed. Magnifications ×200.

FIG. S12 is a group of dynamic pictures of real-time inverted microscopyon one treated MCF7 human breast cancer cell using the “medicinal ionbomb” in cell culture chamber. FIG. S12.1 is the image of a pretreatmentcancer cell. FIG. S12.2 shows acute intracellular dehydration of thetreated cancer cell by 6 seconds after starting the treatment. FIG.S12.3. High edema and damage to cell membrane of the treated cancer cellare seen by 12 seconds after starting the treatment. FIG. S12.4. Thecell membrane is broken and a dead cancer cell is seen by 16 secondsafter starting the treatment. Magnifications ×400.

FIG. S13 is a group of pictures of real-time inverted microscopy. FIG.S13.1 is the picture of untreated cancer cells as the control. FIG.S13.2. Bursting of a highly swollen cancer cell treated with the“medicinal ion bomb.” FIG. S13.3 shows 2 completely destroyed cancercells treated by the “medicinal ion bomb.” One cell on the upper part ofthe picture shows the damaged cell membrane, and a large rupture in thesurface of the cell is seen; the entire cell looks like a rottenpineapple. A cell in the lower part of the picture displays only anoutlook of the cell and its cytoplasm, nucleus, and cellular componentsare lost. FIG. S13.4 are 2 destroyed cancer cells. The upper one haslost upper and lower parts of the cell. The lower part of the secondcell has defected. Microscopically, these pathologic features look likethe sites where an explosion of nuclear bombing occurred. Magnifications×400.

FIG. S14. In vivo distribution of the “medicinal ion bomb” (white lines)surrounding tumor cells in a treated mouse tumor model. FIGS. S14.1 andS14.2 demonstrate 2 tumor sections which are fully filled with the“medicinal ion bomb” solution. FIG. S14.3 displays crystal structures ofNa⁺ and Ca²⁺ salts in intercellular gaps. Magnifications ×1000.

FIG. S15 is a group of pictures of light microscopy on a MC38 mouseadenocarcinoma treated using intratumoral injection with the “medicinalion bomb.” The specimen was taken one day after the treatment. FIG.S15.1. A pretreatment tumor slide as the control. FIG. S15.2 is aspecimen taken from the margin of a treated tumor where there is still asmall area of living cancer tissue, indicating that the treated tumortissue has not been fully filled with the “medicinal ion bomb.” FIG.S15.3 is a mouse tumor model where all tumor tissue is completelykilled, indicating that fully filling a tumor with the “medicinal ionbomb” solution is a necessary factor to kill a cancer. Magnifications×200.

FIG. S16 shows pathological findings of light microscopy in a patientsuffered from maxillary sinus carcinoma. The specimen was taken 3 daysafter the treatment with the “medicinal ion bomb.” FIG. S16.1. All tumortissue is completely killed and associated with apparent inflammatoryreactions and congestion of the treated cancer tissue. Red blood cellsare lining on the inner lumen of the blood vessels and infiltration ofinflammatory cells is seen. Magnification ×400. FIG. S16.2 showsmultiple thromboses in the treated tumor blood vessels. Magnification×200. FIG. S16.3 shows a representative thrombosis in a small vein.Magnification ×400.

Electron Microscopic Findings

SEM disclosed ultrastructural damages of human breast cancer modelstreated with the “medicinal ion bomb.” Destruction of surface of cancercells, and innumerous micro-holes in the membrane of cancer cells wereseen. Membranes of cancer cells were split to pieces or debris. Outerlayer tissues of tumor blood vessels came off. Transverse ruptures ofarterioles or venules in tumor tissue were seen. See FIGS. S17 and S18.

FIG. S17 is a group of pictures of SEM of the cell membranes in a humanbreast cancer model in nude mouse treated with the “medicinal ion bomb.”The specimens were taken 24 hours after the treatment. FIG. S17.1. Anuntreated tumor tissue in which the membranes of cancer cells are notdamaged. FIG. S17.2. Serious damage to the membranes of cancer cellsafter the treatment. FIG. S17.3. Many micro-holes in the membranes ofdead cancer cells are seen. Magnifications ×2000.

FIG. S18 is a group of pictures of SEM of tumor vessels in a humanbreast cancer model in nude mouse treated with the “medicinal ion bomb.”The specimens were taken 24 hours after the treatment. FIG. S18.1 is apicture of tumor vessels in an untreated tumor where all tumor vesselsare lotus-like in shape and the surface of these tumor vessels aresmooth and undamaged. Magnification ×1000. FIG. S18.2 is a picture oftreated tumor tissue which shows the shedding of outer layer tissues oftumor blood vessels. Also, transverse ruptures of many arterioles orvenules in tumor tissue are seen. Magnification ×2000. FIG. S18.3 is adamaged large tumor blood vessel after the treatment with the “medicinalion bomb.” A lot of protein materials and blood cells deposit insideblood vascular lumen. Magnification ×1000.

TEM showed destruction of cell membranes, mitochondria, lysosome, andnucleus of the treated cancer cells. Endothelial cells in the innerlining of tumor vessels were split off from the vascular wall. Mosttreated cancer cells were fragmented into several parts. Interstitialtissue of the treated cancer was smashed. All these pathologic andultrastructural features of treated mouse tumors and human cancer modelslooked like the site of post-nuclear bombing (FIGS. S19 and S20).

FIG. S19 is a group of pictures of TEM of tumor cells in MCF7 humanbreast cancer model in nude mouse treated using the “medicinal ionbomb.” The specimens were taken 24 hours after treatment. FIG. S19.1shows 2 an untreated cancer cells as the control. Magnification ×5000.FIG. S19.2 shows a seriously damaged cancer cell where the membranes ofthe cell and nucleus are lost. The nucleus and chromatins arefragmented, and cytoplasm, mitochondria and other ultrastructuralorganelles are destroyed. Magnification ×5000. FIG. S19.3. A group ofcancer cells show irregular surface covered by the “medicinal ion bomb”(sodium salts and calcium salts, looking like a view of snow). The twodark objects are deformed red blood cells. Magnification ×2000.

FIG. S20 is a group of pictures of TEM of damaged tumor vessels in MCF7human breast cancer models in nude mice treated with the “medicinal ionbomb.” FIG. S20.1 is a small arteriole with damaged blood vascular wall.The seven dark objects are deformed red blood cells. Magnification×2000. FIG. S20.2 discloses ruptures of an arteriole wall, and a damagedendothelial cell is coming off from the vascular wall. Magnification×2000. FIG. S20.3 reveals a damaged venule with many breaks in its wall.Magnification ×2000.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is:
 1. A pharmaceutical composition useful for treatinga cancer, malignant tumor, benign tumor and nonmalignant disease viaadministration by intratumoral injection, the composition comprising, inan aqueous solution, a Na⁺ source providing a Na⁺ concentration fromabout 2.0 M to about 5.5 M in the aqueous solution.
 2. (canceled)
 3. Thepharmaceutical composition of claim 1, further comprising a Ca²⁺ sourceproviding a Ca²⁺ concentration from about 50 mM to about 3.0 M in theaqueous solution.
 4. The pharmaceutical composition of claim 3, whereinthe Na⁺ source is NaCl and the Ca²⁺ source is CaCl₂.
 5. (canceled) 6.The pharmaceutical composition of claim 1, wherein the Na⁺ source is aninorganic salt other than NaCl and the Ca²⁺ source is an inorganic saltother than CaCl₂.
 7. (canceled)
 8. The pharmaceutical composition ofclaim 1, wherein the Na⁺ source is an organic salt having an organiccounter ion and the Ca²⁺ source is an organic salt having an organiccounter ion.
 9. (canceled)
 10. (canceled)
 11. The pharmaceuticalcomposition of claim 3, comprising the maximal effective treatmentconcentration of Ca²⁺.
 12. The pharmaceutical composition of claim 1,wherein the tumor is a benign tumor selected from adenoma, angioma,atheroma, fibroma, lipoma, teratoma, thyroma, cyst, polyp, skin and softtissue neoplasm; breast fibrocystic change, benign prostate hyperplasia,thyroid nodules and hyperthyroidism.
 13. (canceled)
 14. (canceled) 15.The pharmaceutical composition of claim 1, wherein the malignant tumoris selected from cancer and malignant tumor of brain, glioma, thyroidcancer, breast cancer, pancreatic cancer, liver cancer, lung cancer,kidney cancer, colorectal carcinoma, cervical cancer, ovarian carcinoma,cancer and malignant tumor of skin and soft tissue.
 16. A compositionuseful for the preparation of a pharmaceutical composition for treatinga cancer, malignant tumor, benign tumor and nonmalignant disease,comprising Na⁺ and Ca²⁺ in amounts such that when appropriate amount ofwater is added thereto produces a pharmaceutical composition comprisingabout 5.0479 M of Na⁺ and about 250 mM of Ca²⁺.
 17. The composition ofclaim 16, wherein the tumor is a benign tumor selected from adenoma,angioma, atheroma, fibroma, lipoma, teratoma, thyroma, cyst, polyp, skinand soft tissue neoplasm; breast fibrocystic change, benign prostatehyperplasia, thyroid nodules and hyperthyroidism.
 18. (canceled)
 19. Thecomposition of claim 16, wherein the tumor is a malignant tumor selectedfrom cancer and malignant tumor of brain, glioma, thyroid cancer, breastcancer, pancreatic cancer, liver cancer, lung cancer, kidney cancer,colorectal carcinoma, cervical cancer, ovarian carcinoma, cancer andmalignant tumor of skin and soft tissue.
 20. (canceled)
 21. A method fortreating a tumor in a patient, comprising injecting into the tumorlesion a pharmaceutically effective amount of a composition comprisingabout 5.0479 M of Na⁺ and about 250 mM of Ca²⁺.
 22. The method of claim21, wherein the tumor is benign.
 23. The method of claim 22, wherein thebenign tumor is selected from adenoma, angioma, atheroma, fibroma,lipoma, teratoma, thyroma, cyst, polyp, skin and soft tissue neoplasm;breast fibrocystic change, benign prostate hyperplasia, thyroid nodulesand hyperthyroidism.
 24. The method of claim 21, wherein the tumor ismalignant.
 25. The method of claim 24, wherein the malignant tumor iscancer and malignant tumor of brain, glioma, thyroid cancer, breastcancer, pancreatic cancer, liver cancer, lung cancer, kidney cancer,colorectal carcinoma, cervical cancer, ovarian carcinoma, cancer andmalignant tumor of skin and soft tissue.
 26. The method of claim 21,further comprising surgically remove the treated tumor lesion. 27-29.(canceled)
 30. The method of claim 21, wherein the patient suffers fromprimary or recurrent hyperthyroidism.
 31. The method of claim 30,wherein the patient takes iodine by mouth prior to treatment andinjecting comprising performing a longitudinal injection in each thyroidgland under the guidance of an ultrasound imager. 32-39. (canceled)